Role of Transferrin in Iron Metabolism

*Nitai Charan Giri*

#### **Abstract**

Transferrin plays a vital role in iron metabolism. Transferrin is a glycoprotein and has a molecular weight of ~80 kDa. It contains two homologous iron-binding domains, each of which binds one Fe (III). Transferrin delivers the iron to various cells after binding to the transferrin receptor on the cell surface. The transferrintransferrin receptor complex is then transported into the cell by receptor-mediated endocytosis. The iron is released from transferrin at low pH (e.g., endosomal pH). The transferrin-transferrin receptor complex will then be transported back to the cell surface, ready for another round of Fe uptake and release. Thus, transferrin plays a vital role in iron homeostasis and in iron-related diseases such as anemia. In the case of anemia, an increased level of plasma transferrin is often observed. On the other hand, low plasma transferrin level or transferrin malfunction is observed during the iron overdose. This chapter will focus on the role of transferrin in iron metabolism and diseases related to transferrin.

**Keywords:** Transferrin, metabolism, transferrin receptor, homeostasis, endocytosis, intestine, divalent metal transporter (DMT1), Steap3, endosome

#### **1. Introduction**

Iron metabolism is one of the most intricate processes involving many organs and tissues, such as the intestine, the bone marrow, the spleen, the liver, etc. [1, 2]. Various proteins are also involved in maintaining iron homeostasis. Transferrin is a glycoprotein that plays a central role in iron metabolism [3]. It is present at a concentration of 30-60 μM in blood [4]. Transferrin can be divided into several sub-groups – serum transferrin, lactoferrin, and ovotransferrin. Hepatocytes produce serum transferrin found in serum, CSF, etc. Mucosal epithelial cells produce lactoferrin found in milk [5]. Lactoferrin is also found in secretions such as tear and saliva and cells such as neutrophils and leukocytes. Ovotransferrin is an iron-binding protein found in avian egg white. Together transferrins form the most important iron regulation system by transporting iron from the intestine or the sites of heme degradation to proliferating cells [6, 7]. This chapter will focus on the role of transferrin in iron metabolism.

Unlike ferritin, transferrin is a relatively new protein and is found only in phylum Chordata. Transferrin contains ~680 amino acid residues and two subdomain (N-and C-terminal domains) or lobes (**Figure 1**) [8]. It has a molecular weight of 80 kDa.

**Figure 1.** *Structure of human transferrin (top) and two of its iron (brown sphere)-binding sites (bottom).*

The N-terminal lobe consists of residues 1-330 (approx.), while the C-terminal lobe consists of residues 340-680 (approx.) [9]. The two subunits are connected by a small hinge (residues 330-340). Transferrins show high sequence similarity - ~70% identity among lactoferrins while 50-60% identity between lactoferrin and transferrin [10]. The N- and C-terminal halves of these molecules show ~40% sequence identity. It has been suggested that the transferrin molecule may have evolved from the structural gene of an ancestral protein possessing only one metal-binding site and about 340 amino acids by gene duplication [11, 12]. This gene duplication might have led to an increase in its Fe(III) binding capacity and affinity [13]. Although transferrin contains many cysteine residues, it does not have any free sulfhydryl groups (present as disulfide).

Transferrins show greater species variability in carbohydrate composition than in their amino acid composition. The total carbohydrate content varies from 3–12% weight of protein. The number of carbohydrate chains per protein molecule varies from 1 to 4. Human serum transferrin contains about 6% carbohydrate. This carbohydrate moiety has two identical, branched hetero-saccharide chains attached to the amide group of Asn residues via N-glycosidic linkages. However, a minor population of transferrin contains only tri-branched glycans. These carbohydrate groups affect the recognition and conformation of the native protein. The carbohydrate groups can also influence the solubility of the protein.

### **2. Iron binding to transferrin**

Transferrins are bilobal where each lobe reversibly binds a ferric iron (logK = 22.5 for C-site and 21.4 for N-site). Although two iron sites can be distinguished by kinetic and few other studies, their coordination environments are similar (**Figure 1**, bottom). X-ray crystallography indicates that the iron-binding site involves two phenolate oxygen from Tyr, two oxygen from bidentate bicarbonate, nitrogen from His, and oxygen from the carboxylate group of an Asp. Although transferrin binds Fe(III), iron is absorbed as Fe(II) in the intestine. Ceruloplasmin may catalyze the oxidation of Fe(II) to Fe(III) so that it may be bound to transferrin. However, there is some evidence that transferrin binds Fe(II), although with a much lower affinity. The resulting Fe(II)-transferrin-bicarbonate (or carbonate) will be oxidized by molecular oxygen to Fe(III)-transferrin-bicarbonate (or carbonate) [14, 15]. Thus, transferrin binds two Fe(III) in the presence of carbonate or bicarbonate to form a pink-colored complex with an absorption maximum of 465-470 nm. This iron-binding is pH-dependent, where the efficiency of iron-binding is maximum at pH between 7.5 and 10. This ironbinding efficiency decreases upon lowering the pH, and partial dissociation occurs at pH 6.5. Complete dissociation of iron occurs at pH 4.5. This decrease in iron-binding efficiency is useful for preparing apo-transferrin *in vitro*. For every Fe(III) bound to

**Figure 3.** *Distribution of surface charge in apo-transferrin (left) and holo-transferrin (right).*

the protein, three protons are released. Considering that two Tyr residues are bound to Fe(III), these two ligands may be responsible for two protons. The third proton may come from bicarbonate.

Superimposition of the apo-transferrin (no iron) structure with the holo form (diferric) indicates the presence of open and closed forms, respectively (**Figure 2**). It has been suggested that apo-transferrin can exist both in an open and closed formation. However, the closed conformation exists less than 10% of the time [16]. Differential scanning calorimetry experiment performed by titrating Fe(III) into apotransferrin indicated cooperativity between the two lobes in transferrin. During this process, Fe(III) first binds to the C-lobe and then to the N-lobe. It was also observed that the binding of Fe(III) in the C-lobe helps strengthen the binding of Fe(III) in N- lobe. It is worth noting that the interface of the lobes contains hydrophobic patches. The hydrophobic interaction may cause the movement in one lobe as the other one closes due to Fe(III) binding. Also, Fe(III) binding to transferrin alters the surface charge (**Figure 3**). The surface charge in holo-transferrin is more negative than apo-transferrin. Thus, electrostatics may drive the onset of endocytosis.

### **3. Transfer of iron from transferrin to cells**

Under physiological conditions, Fe(III) is tightly bound to transferrin. Considering the Ka of the reaction between transferrin and iron, iron will take thousands of years to dissociate spontaneously from transferrin to blood. Thus, there must be some unique mechanism for transferring iron from transferrin to cells. Now it is established that transferrin receptors on the cell surface play an important role in transferring iron from transferrin to cells. Transferrin receptor is a 180 kDa homodimer type II transmembrane glycoprotein [17]. Two monomers of ~769 amino acids are linked by two disulfide bridges [18]. Each monomer contains three major domains – a C-terminal extracellular domain, a transmembrane domain, and an N-terminal intracellular domain. The C-terminal extracellular domain comprises ~671 amino acids and has two main subunits – domain head and a stock of ~37 amino acids that separate the head from the transmembrane domain. The transmembrane domain consists of ~20-28 residues that create a hydrophobic region. This region contains palmitoylation sites (Cys62 and Cys67) that help the transferrin receptor

*Role of Transferrin in Iron Metabolism DOI: http://dx.doi.org/10.5772/intechopen.100488*

cling to the cell membrane. The intracellular N-terminal domain is made up of ~61-66 residues. Due to the dimeric nature of the transferrin receptor, it binds two transferrin molecules.

#### **3.1 Conformational change in the transferrin receptor due to transferrin binding**

X-ray crystal structure of iron-bound transferrin in complex with transferrin receptor provides the molecular details of the interaction between transferrin and transferrin receptor (**Figure 4**) [19]. Conformation changes in the C-terminal domain of transferrin receptor occurs when transferrin receptor binds iron-bound transferrin. These conformation changes have been suggested to be responsible for initiating endocytosis for Fe(III) uptake by the cells. The most dramatic change in the transferrin receptor structure is observed in the loop containing Asn317 (one of the three glycosylation sites, **Figure 5A**). Also, Phe316 is shifted by ~8 Å while His318 is shifted by ~12.5 Å. These movements bring these residues closer to the C-terminus of other transferrin receptor monomer (**Figure 5B**). The binding of transferrin to transferrin receptor leads to a rotation along transferrin receptor dimeric interface bringing four His (His475 and His684 from each monomer) into proximity (**Figure 5C**). The binding of transferrin to transferrin receptor also causes two Trp residues (Trp641 and Trp740) to undergo significant changes (**Figure 5D**).

The interaction of the transferrin receptor with transferrin also depends on pH. For these interactions, the important pH values are 7.4 (blood pH) and 5.5 (endosomal pH). It has been reported that at blood pH diiron bound transferrin exclusively formed saturated transferrin-transferrin receptor complex [20]. However, monomeric

#### **Figure 4.**

*Interaction of transferrin (cyan: The brown sphere is transferrin-bound iron) with C-terminal extracellular domain of transferrin receptor (green).*

#### **Figure 5.**

*Conformational change of transferrin receptor (cyan) due to the formation of transferrin-transferrin receptor complex (green): Structural change of the loop containing Asn317 (A), movements of Phe316 and His318 from one monomer (indicated by ') to the other monomer (B) four his residues of transferrin receptor come to close proximity due to transferrin binding (C) and movements of Trp641 and Trp740 from one monomer (indicated by ') to the other monomer (D).*

transferrin (N-lobe or C-lobe) with one iron as well as apo-transferrin could not saturate the transferrin receptor. It was shown that the diiron-containing transferrin has the maximum affinity for the transferrin receptor, while the apo-transferrin had the lowest affinity for the transferrin receptor. However, this trend is reversed at endosomal pH. Here, apo-transferrin has the highest affinity for the transferrin receptor. This strong interaction is necessary for transferrin to return to the cell surface and repeat further Fe(III) uptake.

Thus, the recognition of diiron bound transferrin is essential for initiating endocytosis (**Figure 6**). This transferrin-mediated endocytosis involves the clathrin coating of the ensuing endosome to protect it from proteolytic degradation. Thus, the enclosed transferrin and transferrin receptor are protected so that they can be recycled. The adaptor protein complex mediates the formation of a proton-pumping endosome that includes other membrane proteins such as Steap3, a ferrireductase [21, 22]. Once the endosome enters the cell, it is acidified to a pH of 5.5. This acidification may enable the chelator to penetrate the metal-binding site and induce a semi-open conformation that ultimately leads to metal release. It has been reported that the sulfate binding to Fe(III) prevents His and Asp from binding to Fe(III) and thus leads to a

semi-open conformation of the transferrin (**Figure 7**). During the metal release and its delivery to the cytosol via the divalent metal transporter 1 (DMT1), the reduction of Fe(III) to Fe(II) occurs. However, there is some debate about the order of chelation

**Figure 6.**

*Proposed pathway of transferrin receptor mediated endocytosis and iron release into the cytosol.*

#### **Figure 7.**

*Sulfate binding to iron (brown sphere) leads to semi-open conformation by preventing the ligation of his and asp to iron.*

and reduction. One group of researchers think that acidification coupled with chelation by an intracellular chelator (e.g., citrate, ATP) results in the dissociation of Fe(III) from transferrin [23]. This Fe(III) is then reduced to Fe(II) by Steap3. DMT1 then transports Fe(II) into the cytosol, forming a labile iron pool [24, 25]. However, this Fe(II) is quickly stored in ferritin and inserted into various iron-dependent proteins [26, 27]. Another group of researchers believes that the interaction between diiron bound transferrin and transferrin receptor alters the redox potential of Fe(III) from −0.53 V to −0.3 V (vs. SHE) [28, 29]. Then Steap3 will reduce Fe(III) to Fe(II), which

will weaken the metal affinity of transferrin (logβ value of diFe(III) bound transferrin is 43.5 while that of diFe(II) bound transferrin is 13) [30, 31]. Fe(II) will then undergo facile dissociation, possibly with the help of a chelator, and be transported out of the endosome by DMT1. Some researchers also believe that ascorbate is the likely reducing agent [32]. Recently, Fe(III) bound to citrate near the transferrin metal-binding site has been reported (**Figure 8**). However, in this structure, Fe(III) is not ligated to any protein-derived ligand. This citrate-bound iron (without protein-derived ligand) can be considered citrate scavenging of Fe(III) from transferrin.

### **4. Significance of bilobal transferrin**

Since no eukaryotic single-lobed transferrin is known [33], it is reasonable to think that the emergence and persistence of a bilobal structure offer substantial advantages to the organism that uses transferrin for iron transport. However, the nature of the advantages has not been confirmed. One hypothesis is that the bilobal protein resists loss through glomerular filtration in the kidney [34]. However, this hypothesis has been questioned since the bilobal structure may have evolved before the filtration kidney appeared [35]. It has been reported that the C-lobe of full-length transferrin binds iron with four times higher affinity than the isolated C-lobe at pH 7.4 [36]. This affinity becomes 25 times at pH 6.7. Iron release from the N-terminal lobe occurs in the pH range from 6 to 4 compared with 4 to 2.5 for native lactoferrin. These results also support the idea that the more facile iron release from the half-molecule (N-terminal lobe only) compared to the full-length protein is due to the absence of stabilizing interactions between N-terminal and C-terminal halves [10]. It appears that the efficiency of iron release from the C-lobe of native transferrin is impaired by stabilizing interactions of the lobes with each other that retard the release of iron. However, the binding of the transferrin receptor will overcome this problem. Thus, the bilobal structure is favored during evolution so that iron will be released from transferrin when it is complexed with the transferrin receptor.

### **5. Kinetics of iron release from bilobal transferrin**

Although the members of the transferrin family have essentially the same fold due to the high degree of sequence identity, individual transferrin differs in their ironbinding property [37, 38]. Mechanism of iron release from each lobe differs mainly due to the differences in second shell residues [23]. *In vitro* studies with purified transferrin have shown that the iron-loaded protein releases iron as the pH is lowered [39]. Iron-loaded human serum transferrin releases iron over a pH range of 6.5 to 4, whereas the iron release from lactoferrin occurs in the pH range of 4 to 2.5 [40]. Hen serum transferrin releases the first iron in the pH range of 6.5 to 5.2 [41]. Under similar conditions, human serum transferrin 6.0 to 5.5. The loss of the remaining iron from hen serum transferrin C-lobe occurs over a pH range of 5.2 to 4. Studies performed in the absence of transferrin receptor indicate that 96% of the time, iron is released from N-lobe followed by slow release from C-lobe [42]. Also, there is cooperativity among the two lobes in the absence of transferrin receptors [43]. Thus, the iron release from the N-lobe is sensitive to the C-lobe. On the other hand, iron is released from the C-lobe 65% of the time in the presence of the transferrin receptor.

#### **5.1 Iron release from C-lobe**

Iron release from the C-lobe of transferrin is very slow and unaffected by N-lobe [44]. C-lobe has a triad of Lys534-Arg632-Asp634 that controls the iron release in the absence of the transferrin receptor [45, 46]. Lys534 and Arg632 in the C-lobe may share a H-bond that is stabilized by Asp634. Thus, the protonation of Asp634 will trigger the iron release. However, in the structure of pig transferrin, the Lys and Arg are too far away (~4.1 Å apart) to share a H-bond [47]. However, mutation of Lys/ Arg to Ala severely retards iron release from C-lobe [48]. Iron release from the C-lobe in the presence of transferrin receptor proceeds via a different mechanism and is 7-10 fold faster than that in the absence of transferrin receptor. Recent studies have shown that the iron release from the C-lobe is dictated by His349 [49]. Based on the cryo-EM, it was suggested that a pair of hydrophobic residues (Trp641 and Phe760) interact with His349 and stimulates iron release by stabilizing the apo-transferrin/ transferrin receptor complex [50]. The role of His349 in the iron release has been demonstrated by mutating His349 to Ala. In this H349A mutant, the iron release from C-love is reduced by 12 fold.

#### **5.2 Iron release from N-lobe**

In the absence of transferrin receptors, the iron release from N-lobe is controlled by the protonation of a pair of Lys. These two Lys residues are 3 Å apart and share a H-bond [51]. When the pH is reduced, protonation of one of the Lys residues causes the positively charged Lys residues to repeal each other (moving at least 9 Å apart) [52]. This repulsion triggers a cleft opening as well as the release of iron [53]. Mutations of any of these to two Lys to either Glu or Ala drastically slowed the rate of iron release [54]. The release of iron from the N-lobe is further facilitated by the binding of anions to Arg143 [55].

### **6. Stabilization of apo-transferrin/transferrin receptor complex**

The return of apo-transferrin to the cell surface is a distinctive feature of the endocytic cycle. As revealed by the apo-transferrin structure, the N-lobe is stabilized by a salt bridge between Asp240 and Arg678 [56]. Additionally, the PRKP loop (residues 142-145) is connected to the bridge by a disulfide bond (between Cys137 and Cys331). In the apo-transferrin structure, the movement of the PRKP loop and the disulfide bond brings the bridge closer to the protease-like domain of the transferrin receptor to possibly further stabilize the apo-conformation in a pH-dependent manner.

### **7. Biological function of transferrin**

Transferrin delivers the iron to various cells after binding to the transferrin receptor on the cell surface. The transferrin-transferrin receptor complex is then transported into the cell by receptor-mediated endocytosis. The iron is released from transferrin at low pH (e.g., endosomal pH). The transferrin-transferrin receptor complex will then be transported back to the cell surface, ready for another round of Fe uptake and release. This process can turn over roughly a million atoms of iron per cell per minute in active reticulocytes [57]. It is well known that Fe(III) salts are highly

#### *Role of Transferrin in Iron Metabolism DOI: http://dx.doi.org/10.5772/intechopen.100488*

susceptible to hydrolysis at neutral pH to produce insoluble ferric hydroxide. Thus, the concentration of free Fe(III) in physiological fluids will be very low (~10−18 M). However, the daily turnover of hemoglobin iron is ~30 mg (~10−4 M). Thus, there is a need for a high-affinity iron-binding protein, like transferrin. By binding iron upon its release into the bloodstream, serum transferrin prevents the hydrolysis and precipitation of iron [58]. Thus, it increases the solubility of iron in the blood to the micromolar level and consequently increases its bioavailability [59]. Transferrin is usually ~30% saturated with iron with ~27% diferric transferrin, 23% monoferric transferrin (N-lobe), 11% monoferric transferrin (C-lobe), and 40% apo-transferrin [60, 61]. During increasing iron overload, the empty iron binding sites in transferrin are occupied, and thus, iron toxicity is not overserved until transferrin has been saturated with iron. Serum transferrin also inhibits the reduction of Fe(III) to Fe(II), which may lead to iron toxicity via the formation of reactive oxygen species. By having a very high affinity for Fe(III), transferrin can prevent the uptake of Fe(III) by pathogenic microorganisms. The most important role of transferrin is in the transport of iron among the site of absorption (intestinal mucosal cells), utilization (immature erythroid cells), storage (liver), and hemoglobin degradation. Thus, transferrin plays a vital and central role in iron metabolism (**Figure 9**). Although transferrin has a high molecular weight and binds only two iron ions, it is relatively efficient since it is used in many cycles of iron transport. Transferrin is recycled more than 10 times a day to supply the 20-30 mg irons needed for over 2 million erythrocytes produced every second by the bone marrow. Although iron bound to transferrin is <0.1% (4 mg) of total body iron, it constitutes the most critical iron pool with the highest turnover rate (25 mg per day) [62, 63]. It has a relatively longer half-life of 8-10 days *in vivo*. It has been suggested that plasma aluminum, when it binds to transferrin, may lead to anemia since aluminum will enter the iron distribution pathway [64].

**Figure 9.** *A simple diagram of the iron homeostasis in human.*

**Figure 10.**

*Superimposition of structure of transferrin (green) and lactoferrin (cyan). Iron bound to transferrin and lactoferrin is shown as brown spheres.*

In contrast, lactoferrin possesses various biological properties, including antioxidants, antimicrobial and anti-inflammatory activities [65]. Lactoferrin's affinity for iron is very high (double that of transferrin). This high iron affinity partly determines its function. Superimposition of the structure of transferrin and lactoferrin indicates that the lactoferrin has a relatively closed conformation compared to transferrin (**Figure 10**). This closed conformation may explain the higher iron affinity of lactoferrin since once iron is sequestered, it cannot escape. The high affinity of lactoferrin for iron will also enable it to deprive microorganisms of essential metals for growth. However, iron is a crucial nutrient for pathogenic microorganisms which require iron to survive and replicate. Thus, lactoferrin is considered to form part of the immune system since it deprives the pathogenic microorganisms of iron and combats the infection they cause [66]. While the apo-lactoferrin inhibits the growth of a large number of pathogenic bacteria, holo-lactoferrin shows significantly lower inhibition towards these pathogens.

#### **8. Iron sequestration from transferrin by** *N. Meningitidis*

*N. Meningitidis* is a pathogenic bacterium responsible for bacterial meningitis. It acquires iron from transferrin during infection through a transferrin receptor system composed of two proteins TbpA and TbpB [67]. TbpA is a 100 kDa TonB-dependent outer membrane protein required for iron uptake [68]. It can serve as a channel for iron transport across the outer membrane. TbpB is a bilobal protein (60-80 kDa) required for colonization in the host [69]. This protein extends from the outer membrane into

the host milieu to interact with transferrin to initiate iron acquisition [70]. Together, these proteins sequester iron from transferrin. TbpA binds both holo- and apo-forms of transferrin [71]. However, TbpB binds only the holo-transferrin.

#### **Figure 11.**

*Structure of transferrin (green) and transferrin binding protein TbpB (cyan) complex (top: The brown sphere is transferrin-bound iron). Interaction of His349 in transferrin with various residues in TbpB via water (sphere, bottom).*

The crystal structure of TbpB with and without human transferrin has been reported [72]. Both TbpA and TbpB bind to transferrin C-lobe [73]. Within this structure, several amino acids residues of transferrin (His349, Lys511) and TbpB (Arg199, Glu222) are buried in the binding interface (**Figure 11**, top). His349 residue of transferrin interacts with TbpB through a tetrahedrally coordinated water involved in H-bonding with His143, Asp159, and Lys206 from TbpB (**Figure 11**, bottom). Protonation of His349 will cause an electrostatic repulsion with Lys511 leading to a conformational change which results in an open conformation of transferrin. Thus, His349 in human transferrin can act as a pH-inducible switch for iron release in the presence of the transferrin receptor [19, 49]. Structure-based pKa prediction suggests that TbpB binding to human transferrin leads to a reduction of the estimated pKa of human transferrin His349 from 6.2 (unbound form) to 1.9 (bound to TbpB) [74]. Thus, the binding of TbpB to transferrin may prevent His349 from becoming protonated and stabilize the holo C-lobe of transferrin. Therefore, TbpB does not initiate the opening of the human transferrin holo C-lobe that results in the iron release. Thus, the first step of iron acquisition by TbpB will involve binding to iron-loaded transferrin inside the host and maintaining the iron-loaded form until its delivery to TbpA in the second step.

Although the human Transferrin receptor interacts with transferrin via both N and C lobes, TbpB interacts with only the C-lobe of transferrin. Also, the human transferrin receptor binds to both apo- and holo-forms of transferrin, while TbpB binds specifically to holo-transferrin [75]. This is because the holo-form of C-lobe with a closed conformation will allow more effective docking of C-lobe of transferrin onto TbpB. However, the apo form of transferrin with an open structure will drastically reduce the binding interface between TbpB and transferrin. Unlike human transferrin receptor, TbpB interacts with a loop (residues 496-515) of human transferrin. Variation of this loop is observed among mammalian transferrins. This variation in the TbpB recognition site on transferrin seems to act as the barrier for cross-species specificity between TbpB and transferrin. Finally, bacterial transferrin binding protein (TbpB) competes with the human transferrin receptor for transferrin/iron. TbpB binding site on human transferrin partially overlaps with the transferrin receptor binding site. This overlapping binding site of the human transferrin receptor and pathogenic transferrin binding protein (e.g., TbpB) allows the pathogens to circumvent the mutation of transferrin.

#### **9. Conclusion**

This chapter highlights the role of transferrin in iron metabolism, including the iron-binding by transferrin, transferrin receptor-mediated endocytosis of transferrin/transferrin-receptor complex, and consequent iron release, etc. Some of the released iron will be stored in ferritin, while the other part will be used by various proteins and enzymes constituting a pathway of iron regulation. This iron-binding and regulation by transferrin are critical considering the toxicity of iron. How the differences (amino acids, structure, iron-binding affinity, etc.) between serum transferrin and lactoferrin dictates their biological functions have been highlighted. Finally, the competition between the human transferrin receptor and bacterial transferrin binding protein (e.g., TbpB) for getting iron from transferrin has also been discussed. Understanding the interaction of transferrin with other proteins (e.g., transferrin binding protein from various pathogenic bacteria) may lead to drug development. Overall, elucidation of the role of transferrin in iron metabolism will help understanding iron-related diseases and improve treatment.

*Role of Transferrin in Iron Metabolism DOI: http://dx.doi.org/10.5772/intechopen.100488*

### **Author details**

Nitai Charan Giri Central Institute of Petrochemical Engineering and Technology, Raipur, India

\*Address all correspondence to: nitaigiri@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## **Chapter 4** Hepcidin

## *Safa A. Faraj and Naeem M. Al-Abedy*

### **Abstract**

The hepcidin is antimicrobial peptide has antimicrobial effects discover before more than a thousand years; it has a great role in iron metabolism and innate immunity. Hepcidin is a regulator of iron homeostasis. Its production is increased by iron excess and inflammation and decreased by hypoxia and anemia. Iron-loading anemias are diseases in which hepcidin is controlled by ineffective erythropoiesis and concurrent iron overload impacts. Hepcidin reacts with ferroportin. The ferroportin is found in spleen, duodenum, placenta, if the ferroportin decrease, it results in the reduced iron intake and macrophage release of iron, and using the iron which stores in the liver. Gene of human hepcidin is carried out by chromosome 19q13.1. It consists of (2637) nucleated base. HAMP gene was founded in the liver cells, in brain, trachea, heart, tonsils, and lung. Changing in the HAMP gene will produce a change in hepcidin function. The hepcidin is made many stimulators are included opposing effects exerted by pathological and physiological conditions. Hepcidin is essential for iron metabolism, understanding stricter and genetic base of hepcidin is crucial step to know iron behavior and reactions to many health statuses.

**Keywords:** hepcidin, iron, HAMP gene

#### **1. Introduction**

The hepcidin is antimicrobial peptide has antimicrobial effects discover before more than a thousand years; it has a great role in iron metabolism and innate immunity [1]. It's a peptide hormone produced by the liver that acts as an iron regulator. Hepcidin is an iron homeostasis regulator. Iron deficiency and inflammation boost its production, while hypoxia and anemia diminish it. Hepcidin prevents iron from duodenal enterocytes absorbing dietary iron, macrophages recycling iron from senescent erythrocytes, and iron-storing hepatocytes from entering the bloodstream. Hepcidin is controlled by inefficient erythropoiesis and concurrent iron overload consequences in iron-loading anemias [2].

Human urine and blood, particularly plasma after filtration, were used to isolate hepcidin. Macrophages, adipocytes, neutrophils, lymphocytes, kidney cells, and -cells all make hepcidin. The studies experiment on mice used for determination hepcidin regulation, expression, function, and structure. Severe iron overload is occurring due to the gene responsible with hepcidin production; the gen has the role of iron function. However, decreased and iron increased hepcidin expression in transgenic animals. Hepcidin serves a variety of purposes, including inflammation, hypoxia, and iron storage [3].

Ferroportin reacts with hepcidin. The ferroportin is located in the spleen, duodenum, and placenta; a decrease in ferroportin results in reduced iron intake and iron release by macrophages, as well as the use of iron stored in the liver [4].

### **2. The** *HAMP* **gene and structure of hepcidin**

The human hepcidin gene is located on chromosome 19q13.1. It is made up of 263 nucleated bases. The HAMP gene was discovered in liver cells, brain cells, trachea, heart, tonsils, and lung cells [5].

Hepcidin comes in three forms: 25 aa, 22 aa, and 20 aa peptide. The HAMP gene encodes preprohepcidin, which has 84 amino acids. The structure of hepcidin25 (**Figure 1**), which consists of (8) cysteine linked by a disulfide bond, is detected in urine, while 25 and 20 are found in human serum. The structure of hepcidin is studied using NMR spectroscopy; it has four disulfide links [7].

#### **Figure 1.**

*Molecule structure of human synthetic hepcidin-25. Background: Hepcidin-25. Front: Showing the general structure of hepcidin-25. Gray arrows are distorted* β*-sheets, and colored gray is peptide backbone. Colored yellow is a disulfide bond, blue is indicate to positive residues of lysine and arginine, red indicates to the negative residue of aspartic acid, and colored green indicates to histidine which containing amino-terminal [6].*

#### **3. Hepcidin gene regulation**

Location of the HAMP gene is at 19q13 chromosome mRNA. Several genetic factors affect on iron concentration, hypoxia, inflammation, erythropoiesis, and anemia. All these factors have two pathways on the gene. The first signaling is by bone proteins and the second Janus kinase/signal related to inflammation [8].

The protein is regulated of hepcidin level depend on transferrin and interaction receptor. HFE is chanced from TfR1 [Tf-Fe3+] to promote its interaction with (TfR2).

#### *Hepcidin DOI: http://dx.doi.org/10.5772/intechopen.101591*

TfR2 and HFE link with the receptor of hemojuvelin by the BMP/Son for activating HAMP gene. This reaction stimuli phosphorylation of BMP receptor, and stimulating signals into the cell. The receptor of type II activates receptor of type I, then the signal transmits to the SMAD regulatory receiver, phosphorylating (SMAD-8, SMAD-5, and SMAD-1). The activated complex transfer to the nucleus for regulating gene transcription [9]. Matriptase-2 protein and SMAD-4 is a suppressor of BMP/SMAD. HJV is reacting with Matriptase-2 and causes fragmentation. Growth hormone and erythropoietin associate with the receptor, wherever, interferon and cytokines. The hepcidin wad produced in the liver, it increases if iron gets in liver cells. Hepcidin creates and released into the blood vessels and spread all the body. It interacts with other proteins in the liver, intestines, and WBC for iron storage when the hepcidin was produced at large amounts, increases the occurrence of liver tumor or chronic or acute hypoferremia. If the hepcidin is decreased in the production, results in mutations in the hemojuvelin gene, hepcidin gene, or *transferrin receptor 2* [10].

### **4.** *HAMP* **gene mutation**

Hepcidin function will be altered if the HAMP gene is altered. Exon 3 of the HAMP gene encodes proteins, and it is regarded the most critical and biggest section of the gene, containing several polymorphisms [10]. The HFE gene has more polymorphisms than the HAMP gene. There are approximately 16 forms of single nucleotide polymorphism that have been discovered in various investigations [8]. Mutations in the gene have been reported in a number of reports. People who have mutations in the HAMP gene develop juvenile hemochromatosis between the ages of 10 and 30. As the initial genetic change in the HAMP gene, microsatellite marker probes are utilized. After exchanging several amino acids in the active peptide, or replacing C78 with a tyrosine, C78T, the mutation occurs in c.233G > A at some point [11].

The mutation allows ferroportin to form bisulfite connections with hepcidin, resulting in an increase in iron absorption. C70R mutations result in cysteine bisulfite bond distortion. The arginine replaces the cysteine, which does not allow the creation of the bisulfite bridge between 3 and 6 in the hepcidin peptide. C to T substitutions were found at position (166) of the HAMP (166C-T), as well as arginine substitutions at position (56) for a halting codon (R56X), 193A to R56X. (T). Furthermore, the ferroprotein does not bind to hepcidin, resulting in the production of additional iron. In contrast, deleting guanine from exon two at location 93 causes an RNA mutation. The deletion of Met50del IVS21 from exon two causes a disruption in the active peptide's expression as well as variations in reading frames. Met50 and (IVS + 1 (G)) are suppressed by the mutation. The reading frame is lengthened as a result of this mutation. Another mutation, G71D, causes a change in amino acid 71, which lies between 3 and 4 cysteine and precludes ferroprotein binding. In sickle cell disease patients, the HFE-H63D mutation is linked to the HAMP-G71D variant, which increases iron overload [12].

The polymorphism (G to A) occurs at the +14 position of the 5′-UTR region, resulting in a new initiation codon, a new aberrant protein, and a shift in the reading frame. After the mRNA is translated, an unstable protein will be produced, which will be analyzed. The related polymorphisms NC-582A > G and NC-1010C > T in the HAMP gene create a haplotype with ferritin concentrations greater than 300 g/L [13].

HFE gene polymorphisms are frequently linked to HAMP. With iron overload, there are various mixed clinical symptoms in some clinical instances. The variations C, 582A > G and C-153C > T reduce hepcidin expression, but the peptide's mode of action remains same without transferrin saturation and increased ferritin levels. The patient who has HAMP gene mutations were cannot make hepcidin and unable to decrease iron absorption. The body organs become contain iron at large amounts such as heart and liver, and it will affect with damage. Any change in the HAMP gene could result in a faulty hepcidin protein, and it would have no effect. The accumulation of iron and ferritin in the organs contributes to the development of diseases in several organs, such as coronary artery disease, diabetes mellitus, HIV, HBV, and HCV, where reactive oxygen generates oxidative material that damages tissues. And some neurological illnesses, such as Alzheimer's, Parkinson's, and sclerosis, are linked to high levels of hepcidin in the blood [14].

#### **5. The hepcidin clinical applications**

The hepcidin is made many stimulators are included opposing effects exerted by pathological and physiological conditions. The response is usually rapid. The hepcidin production increases during few hours after inflammatory stimulation and iron administration. Several stimuli could associate with hepcidin. Such as in hepcidin production and severe ID with the inflammation [15, 16].

Several ineffective conditions, such as signals released by bone marrow and non-transfusion-dependent thalassemia. The results showed hepcidin suppression non-transfusion-dependent thalassemias other iron-loading anemias, and even in-thalassemia trait. Serum hepcidin in transfusion-dependent b-thalassemia showed increasing in blood transfusions and decreasing through inter-transfusion periods [16].

Clinically relevant conditions include CKD, RBC transfusions, iron administration, replete iron stores, TMPRSS6 variants, infections/inflammatory disorders, ineffective erythropoiesis, hypoxia, erythropoietic stimulating agent administration, chronic liver diseases, alcohol abuse, HCV, hemochromatosisrelated mutations, and testosterone estrogen administration. HCV, hereditary hemochromatosis; HH, iron deficiency; IDA, RBC, transmembrane protease serine 6, matriptase-2 encoding gene; CKD, glomerular filtration rate; GFR, hepatitis C virus; HCV, hereditary hemochromatosis; IDA, RBC, transmembrane protease serine 6, and matriptase-2 encoding [17].

#### **6. Structure and location of** *HFE* **gene human**

HFE protein was encoded by the HFE gene in humans. The gene lies at chromosome six—6p21.3. The protein is included membrane protein such as MHC class I-type and link with beta-2 microglobulin. HFE protein regulates iron uptake by transferrin HFE protein and the transferrin receptor which composed from (343) amino acid (**Figure 2**). Many other types of proteins such as a signal peptide, transferrin receptor-binding region, and immunoglobulin molecules. HFE is prominent in small intestinal absorptive cells, epithelial cells in stomach, macrophages, and granulocytes and monocytes [19].

#### **Figure 2.**

*The HFE gene diagram. The image was changed after getting permission from the author. Cys282 -> Tyr282 exchanging mutation of C282Y and His63 -> Asp63 exchanging mutation of H63D [18].*

### **7. Maintaining iron homeostasis by hepcidin**

Hepcidin is regulated iron absorption. Pathway of iron is showed in (**Figure 3**), FPN1link with hepcidin is results in iron retention into the cell and do not allow of iron from getting in the plasma. Hepcidin is made and store in the liver cell [20].

**Figure 3.** *Hepcidin internalization and degradation.*

Ferroportin binds to hepcidin results in degradation, wherever the reaction between ferroportin and hepcidin regulates and control on iron concentration. The hepcidin regulation is a very complex mechanism and depends on many transmembrane proteins. JAK-STAT activated HAMP expression in interleukin-6 (IL6) status and inflammation-mediated response [21].

Bone-morphogenetic protein is work as a key for the regulation of *HAMP* gene through *SMAD* signaling pathway.

Also, hemojuvelin is protein made in a lever membrane cell. If the iron becomes low, the hemojuvelin is activated wherever the *sHJV* inhibits *HAMP level* through lining with *BMP*. Regulation of iron is not understood although many proteins work to iron regulation we know it such as (*TFR2*) have great role in iron regulation [22].

#### **8. Iron regulation by hepcidin**

The function of the enterocytes has absorbed the iron, the iron store in the macrophages and hepatocytes and the process is controlled by hepcidin, the hepcidin is produced in the liver. Hepcidin consists of (84) amino acid, it is undergoing for several reactions to become (60) amino acid then transfer to (25) amino acid [23].

The hepcidin is hormone consist of four disulphide bonds and 32% beta-sheet. The function of the hepcidin is control on iron efflux by ferroportin wherever; the liver secretes iron in the plasma. After secretion, the hepcidin is bound with ferroportin; wherever, ferroportin is protein on the cell surface have transferred the iron inside the cell. If the ferroportin is reduced in expression, become the intracellular iron is less. Hepcidin is absorbed iron from the food and transfers to plasma, and the

**Figure 4.** *Regulation of iron balance.*

#### *Hepcidin DOI: http://dx.doi.org/10.5772/intechopen.101591*

iron gets in the cell by binding hepcidin and ferroportin. Reduction of the ferroportin on the cell surface is mechanism unclear [9].

**Figure 4** show how iron flows into plasma exclusively through the membrane channel, ferroportin. Macrophages, enterocyte's hepatocytes are the principal cell types that express ferroportin and so export iron. The duodenum, spleen, and liver, which contain these cells, are important locations for controlling iron flux (blue arrows). Hepcidin, a 25-amino-acid hepatic hormone, regulates ferroportin levels. Endocytosis and proteolysis are triggered when hepcidin binds to ferroportin, preventing iron flow (red arrows) into the plasma from ferroportin-expressing tissues. Hepcidin production rises as iron stocks rise during infection (black arrows) and falls as erythropoiesis demands more iron (red arrows) [24].

### **9. Conclusion**

Hepcidin is essential for iron metabolism, understanding stricter and genetic base of hepcidin is crucial step to know iron behavior and reactions to many health status. This chapter highlights on hepcidin structure and genetic information as well as its relation to iron metabolism.

### **Author details**

Safa A. Faraj1,2\* and Naeem M. Al-Abedy3

1 Department of Pediatrics, College of Medicine, Wasit University, Kut, Iraq


\*Address all correspondence to: safaafaraj@uowasit.edu.iq

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[7] Huang T, Gu W, Wang B, Zhang Y, Cui L, Yao Z, et al. Identification and expression of the hepcidin gene from

brown trout (*Salmo trutta*) and functional analysis of its synthetic peptide. Fish & Shellfish Immunology. 2019;**87**:243-253

[8] Fleming RE, Sly WS. Hepcidin: A putative iron-regulatory hormone relevant to hereditary hemochromatosis and the anemia of chronic disease. Proceedings of the National Academy of Sciences. 2001;**98**(15):8160-8162

[9] Fillebeen C, Charlebois E, Wagner J, Katsarou A, Mui J, Vali H, et al. Transferrin receptor 1 controls systemic iron homeostasis by fine-tuning hepcidin expression to hepatocellular iron load. Blood. 2019;**133**(4):344-355

[10] Pandey S, Pandey SK, Shah V. Role of HAMP genetic variants on pathophysiology of iron deficiency anemia. Indian Journal of Clinical Biochemistry. 2018;**33**(4):479-482

[11] Arts HH, Eng B, Waye JS. Multiplex allele-specific PCR for simultaneous detection of H63D and C282Y HFE mutations in hereditary hemochromatosis. Journal of Applied Laboratory Medicine. 2018;**3**(1):10-17

[12] Rahman HA, Abou-Elew HH, El-Shorbagy RM, Fawzy R, Youssry I. Influence of iron regulating genes mutations on iron status in Egyptian patients with sickle cell disease. Annals of Clinical and Laboratory Science. 2014;**44**(3):304-309

[13] Moreira AC, Neves JV, Silva T, Oliveira P, Gomes MS, Rodrigues PN. Hepcidin-(in) dependent mechanisms of iron metabolism regulation during infection by Listeria and Salmonella. Infection and Immunity. 2017;**85**(9): e00353-e00317

#### *Hepcidin DOI: http://dx.doi.org/10.5772/intechopen.101591*

[14] Go HJ, Kim CH, Park JB, Kim TY, Lee TK, Oh HY, et al. Biochemical and molecular identification of a novel hepcidin type 2-like antimicrobial peptide in the skin mucus of the pufferfish *Takifugu pardalis*. Fish & Shellfish Immunology. 2019;**93**:683-693

[15] Chawla LS, Beers-Mulroy B, Tidmarsh GF. Therapeutic opportunities for hepcidin in acute care medicine. Critical Care Clinics. 2019;**35**(2):357-374

[16] Hanudel MR, Rappaport M, Chua K, Gabayan V, Qiao B, Jung G, et al. Levels of the erythropoietinresponsive hormone erythroferrone in mice and humans with chronic kidney disease. Haematologica. 2018;**103**(4): e141

[17] Lehtihet M, Bonde Y, Beckman L, Berinder K, Hoybye C, Rudling M, et al. Circulating hepcidin-25 is reduced by endogenous estrogen in humans. PLoS One. 2016;**11**(2):e0148802

[18] Swinkels DW, Girelli D, Laarakkers C, Kroot J, Campostrini N, Kemna EH, et al. Advances in quantitative hepcidin measurements by time-of-flight mass spectrometry. PLoS One. 2008;**3**(7):e2706

[19] Yin X, Chen N, Mu L, Bai H, Wu H, Qi W, et al. Identification and characterization of hepcidin from Nile Tilapia (*Oreochromis niloticus*) in response to bacterial infection and iron overload. Aquaculture. 2022;**546**:737317

[20] Aschemeyer S, Qiao BO, Stefanova D, Valore EV, Sek AC, Ruwe TA, et al. Structure-function analysis of ferroportin defines the binding site and an alternative mechanism of action of hepcidin. Blood. 2018;**131**(8):899-910

[21] Wrighting DM, Andrews NC. Iron homeostasis and erythropoiesis. Current Topics in Developmental Biology. 2008;**82**:141-167

[22] Corengia C, Galimberti S, Bovo G, Vergani A, Arosio C, Mariani R, et al. Iron accumulation in chronic hepatitis C: Relation of hepatic iron distribution, HFE genotype, and disease course. American Journal of Clinical Pathology. 2005;**124**(6):846-853

[23] Tangudu NK, Alan B, Vinchi F, Wörle K, Lai D, Vettorazzi S, et al. Scavenging reactive oxygen species production normalizes ferroportin expression and ameliorates cellular and systemic iron disbalances in hemolytic mouse model. Antioxidants & redox signaling. 2018;**29**(5):484-499

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#### **Chapter 5**

## Abnormal Iron Metabolism and Its Effect on Dentistry

*Chinmayee Dahihandekar and Sweta Kale Pisulkar*

#### **Abstract**

Iron is a necessary micro-nutrient for proper functioning of the erythropoietic, oxidative and cellular metabolism. The iron balance in the body adversely affects the normal physiologic functioning of the body and structures in the oral cavity. Various abnormalities develop owing to improper iron metabolism in the body which reflects in the oral cavity. The toxicity of iron has to be well understood to immediately identify the hazardous effects which arise owing to it and to manage it. It has been very well mentioned in the chapter. The manifestations of defects of iron metabolism in the oral cavity should be carefully studied to improve the prognosis of the treatment of the same. Disorders related to iron metabolism should be managed for improvement in the quality of life of the patient.

**Keywords:** iron metabolism, anaemia, iron toxicity, manifestations in oral cavity

#### **1. Introduction**

For optimal erythropoietic function, oxidative metabolism and cellular immunity, iron is required. Cellular iron overload induces toxicity and cell death by producing free radicals and oxidising lipids, both of which are required for cellular metabolism and aerobic respiration. Due to the lack of active iron excretory mechanisms, dietary iron absorption (12 mg/day) is tightly regulated and closely balanced against iron loss. Dietary iron is found in two forms: haem (10%) and nonhaem (ionic, 90%), and both are absorbed in the apical surface of duodenal enterocytes through different mechanisms. Iron is exported via Ferroportin 1 (the only one). Absorbed iron crosses the enterocyte's basolateral membrane into the circulation (possible iron exporter), where it binds to transferrin and is transported to utilisation and storage sites transferrin-bound iron enters target cells via receptor-mediated endocytosis, mostly erythroid cells but also immune and hepatic cells. Senescent erythrocytes are phagocytosed by reticuloendothelial system macrophages; haem is metabolised by haem oxygenase, and the freed iron is stored as ferritin. Later, iron from macrophages will be exported and transferred to transferrin. The erythropoiesis demands (20e30 mg/day) need this internal iron cycle. When transferrin becomes saturated in ironoverload scenarios, excess iron is transported to the liver, the other principal storage organ for iron, creating a risk of free radical generation and tissue damage [1].

The fact that iron's redox pair (Fe(II)/Fe(III) may have potentials varying from −300 to 700 mV, depending on the nature of the ligands and the surrounding environment, contributes to its use. Iron is abundant on the planet's surface; however, it is relatively inaccessible. This is an important aspect of iron metabolism. At neutral pH and in an oxidising environment, iron exists in the three valence state, which is seen in many common microbial environments. As a result, it is extremely difficult to dissolve. The presence of iron storage and transport proteins such as ferritin (FTN), lactoflavin (LFT) and lactoflavin (LFT) limits the amount of iron available to a microorganism residing in an animal host (LFT). Despite the fact that extremely low iron concentrations of 1 mmol (5) are usually sufficient for optimal growth yields, bacteria frequently find themselves in iron-deficient environments and must waste a significant amount of energy to acquire this metal. It is also worth mentioning that bacteria can become iron-overloaded, necessitating careful monitoring of iron intake [1, 2].

Increased iron demands, insufficient external supply and increased blood loss can contribute to iron deficiency (ID) and iron deficiency anaemia. An overabundance of hepcidin hinders iron absorption and recycling in chronic inflammation, leading to hypoferremia and iron-restricted erythropoiesis (functional iron deficiency), and finally, anaemia of chronic illness (ACD), which can advance to ACD with real ID (ACD + ID). Hereditary haemochromatosis (HH type I, caused by mutations in the HFE gene) and hereditary haemochromatosis (HH type II, caused by mutations in the hemojuvelin and hepcidin genes) can both be caused by low hepcidin expression. Changes in the transferrin receptor 2 generate HH type III, whereas mutations in the ferroportin gene induce HH type IV. All of these illnesses show signs of iron excess. In iron overload scenarios, non-transferrin bound iron develops when transferrin becomes saturated. A part of this iron (labile plasma iron) is very reactive, leading to the generation of free radicals. Free radicals induce the parenchymal cell damage associated with iron overload disorders [3].

The teeth, gingiva, oral tissues and muscles are all affected by these major metabolic anomalies of iron metabolism. These processes influencing the oral cavity must be well understood in order to block future advancement and create a comprehensive rehabilitation approach for such persons, taking into consideration the numerous consequences of improper iron metabolism [4].

#### **2. Iron metabolism**

#### **2.1 Iron uptake**

Owing to certain specific mechanisms (as explained in **Figure 1**): (1) transport mechanisms were not required for iron absorption until relatively late in evolution when the environment became oxidising and iron became insoluble, and (2) a range of sources can function as iron providers, bacterial iron assimilation happens via a variety of routes. Many bacteria, in addition, have numerous iron absorption mechanisms. This allows them to acquire iron from a variety of settings and sources. Bacteria can get iron from a number of sources, but regardless of where it comes from, it must be delivered to the cytoplasm through numerous microbial surface layers. An outside membrane, a peptidoglycan layer, and an innermost intracellular membrane are the minimum layers for Gram-negative bacteria. The periplasm, or gap between the outer and inner membranes, is where the peptidoglycan cell wall is found. Gram-positive cells, on the other hand, may only have an exterior peptidoglycan cell wall that is thick and strongly cross-linked. On the basis of the iron source

*Abnormal Iron Metabolism and Its Effect on Dentistry DOI: http://dx.doi.org/10.5772/intechopen.104502*

#### **Figure 1.**

*Gram-negative bacteria's generalised high-affinity iron transport mechanism.) The three fundamental components are shown: (a) an outer membrane receptor protein; (b) a TonB system for activating the receptor protein; and (c) a cytoplasmic membrane-based periplasmic binding protein-dependent ABC transporter. OM stands for outer membrane; PG is for peptidoglycan; and CM stands for cytoplasmic membrane.*

and the manner in which iron is mobilised, a wide range of iron transport systems may be differentiated, although they all follow a similar pattern. Passage across the outer membrane for iron complexed to a carrier requires the presence of an outer membrane receptor protein with a syntactic domain identical to that of the iron complexed to a carrier and is iron-controlled. A receptor protein is specialised for and binds to a certain iron-carrier complex, and it is occasionally generated in large quantities only when that iron complex is accessible [5].

Second, the cytoplasmic membrane proteins TonB, ExbB and ExbD are required for iron entry into the periplasm, whether it is complexed or free. Members of the ABC super transporter family are also engaged in cytoplasmic membrane transport. The transport components, in this case, include a peripheral cytoplasmic membrane, ATPase with two copies and a distinct ATP-binding site motif, as well as two hydrophobic cytoplasmic membrane proteins. In summary, an outer membrane receptor protein, a TonB system and an ABC transporter are required for iron entrance into the cytoplasm of Gram-negative bacteria. The proton-motive force and ATP, respectively, are required for passage across the outer and cytoplasmic membranes. TonB systems have broad specificity, whereas ABC transporters recognise several iron complexes if they are physically related. Outer membrane receptor proteins bind just one particular iron complex, whereas TonB systems have broad specificity [6].

The synthesis and secretion of tiny (600–1000 Da) iron-chelating molecules known as siderophores is a significant method by which bacteria acquire iron. Siderophores are made up of ordinary amino acids, nonprotein amino acids, hydroxy acids, and their production does not need ribosomes despite the presence of amide bonds. Instead, a thiotemplate technique is used, which is quite similar to the one used to make some peptide antibiotics. The mechanism of iron release from siderophores is unknown. Free siderophores, or modified forms, are discharged into the medium when ferrisiderophores enter the cytoplasm. Enzymatic reduction of iron is considered to be the release mechanism since siderophores: (1) bind Fe(II) less readily than Fe(III); and (2) the cytoplasm is a reducing environment [7].

Microbes that can live in oxygen-depleted habitats, such as swamps, intestines and marshes, or acidic environments, where reduced iron is stable and soluble, benefit from the ferrous iron transfer. Fe(II) may enter the periplasm through holes in the outer membrane, and bacteria can transport it through the inner membrane through a number of mechanisms. Some of these cytoplasmic membrane transporters have a broad transition metal selectivity but just a weak affinity for ferrous iron. There are, however, systems that exclusively work with Fe(II) as a substrate. The feo operon encodes one such mechanism that is important in certain bacteria (feoABC) [8]. Members of the OFeT (oxidase-dependent iron transporter) family, which were initially discovered in lower eukaryotes, are another widely dispersed group of Fe(II) transporter proteins. Finally, certain aerotolerant bacteria, such as the Gram-positive *Streptococcus mutans*, acquire iron by converting surface-bound Fe(III) to Fe via a reductase that is exposed on the cell surface (II). The iron is subsequently delivered to the cytoplasm by a ferrous ion transporter. The ABC type of ferric iron acquisition mechanism is found in a variety of Gram-negative taxa, including Serratia, where it was identified and named Sfu type transport. Fe(III) is accepted by a periplasmic binding protein, which then delivers it to the transporter's cytoplasmic components, which internalise the iron. Uptake systems with outer-membrane components can also work in tandem with ferric iron transporters [9].

The bulk of iron in animals is found intracellularly in the form of heme (Hm). Hm, in turn, is a prosthetic group of proteins that includes haemoglobin (Hb), myoglobin and Hm-containing proteins like cytochromes. Iron assimilation routes that detect free Hm are similar to those that identify iron–siderophore complexes; they need (1) a TonB-dependent outer-membrane receptor protein; and (2) an ABC transporter for cytoplasmic membrane crossing [10]. Hm can be removed from Hb by a variety of genera. TonB-dependent Hb-binding proteins are found in the outer membranes of Neisseria and Haemophilus spp. Surprisingly, both of these taxa contain additional TonB-dependent receptors that let them get iron from Hb–Hp complexes. These Hb–Hp receptors might be made up of two distinct proteins. *Serratia marcescens* has a unique mechanism for the first steps in getting iron from Hm or Hb. This bacterium secretes a tiny protein (HasA) that acts as a hemophore via an ABC transporter (Hbp). Only Haemophilus strains have been shown to use Hm in conjunction with hemopexin. The mechanism is not fully understood, but it appears that three genes are necessary, one of which appears to encode a big secreted Hbp (HxuA). HxuA binds hemopexin, removes it, and transports it to an outer-membrane receptor [11].

Iron trafficking exemplifies the cycle economy. During erythrocyte phagocytosis, the majority of iron (20–25 mg/day) is recycled by macrophages; only 1–2 mg of iron is absorbed daily in the stomach, compensating for a loss of the same amount (**Figure 2**) [13]. The duodenum is the location of controlled non-heme iron uptake; nonheme iron is imported from the lumen via the apical divalent metal transporter 1 after duodenal cytochrome B reductase converts ferric to ferrous iron (DCYTBH) (DMT1). There are no known mechanisms by which heme iron absorbs more than non-heme iron. Non-utilised iron in enterocytes is either retained in ferritin (and lost by mucosal shedding) or exported to plasma through basolateral membrane ferroportin (and lost with mucosal shedding) [14].

Iron availability influences the expression of genes that code for proteins required for high-affinity iron absorption. Fur is a crucial regulatory protein found in most Gram-negative and Gram-positive bacteria with low GC content DNA. Fur is an Apo-repressor, a short histidine-rich polypeptide that binds DNA in the presence of

#### *Abnormal Iron Metabolism and Its Effect on Dentistry DOI: http://dx.doi.org/10.5772/intechopen.104502*

its corepressor Fe(II). Fur's negative regulation of genes does not fully explain iron's regulatory actions. Although Fur represses most iron-regulated genes under iron-rich environments, some are positively controlled by Fur, and others are only activated by iron in the absence of Fur (**Figure 3**) [15].

#### **Figure 2.**

*On the luminal side of the enterocyte, the metal transporter DMT1 takes up ferrous iron that has been reduced by DCYTB. After ferrous iron is oxidised to ferric iron by hephaestin, iron not utilised inside the cell is either stored in ferritin (FT) or exported to circulating transferrin (TF) by ferroportin (FPN) (HEPH). Local hypoxia stabilises hypoxia-inducible factor (HIF)-2, which promotes the expression of the apical (DMT1) and basolateral (FPN) transporters. Heme is transformed to iron by heme oxygenase once it enters the cell by an unknown process [12].*

#### **Figure 3.**

*Main iron metabolism routes in animals (based on Munoz et al.2). Key: 1, ferrireductase; 2, divalent metal transporter (DMT1); 3, haem protein carrier 1 (HPC1); 4, haem oxygenase; 5, haem exporter; 6, ferroportin (Ireg-1); 7, hephaestin/caeruloplasmin; 8, transferrin receptor-1 (TfR1); 9, transferrin receptor-1 (TfR1) complex; 10, natural resistance macrophage protein-1 (Nramp-1); 11, mitoferrin; 12, mitochondrial haem exporter (Abcb6); 13, others: bacteria, lactoferrin, haemoglobinehaptoglobin, haemehaemopexin, and so on; 14, caeruloplasmin; 15, transferrin receptor-2 (TfR2).*

#### **2.2 Iron distribution**

Transferrin binds to iron in the bloodstream and distributes it to storage and use sites. Only 30–40% of transferrin's iron-binding capacity is used in ordinary physiological circumstances; hence, transferrin-bound iron is only w4 mg, yet it is the most significant dynamic iron pool. Transferrin-bound iron penetrates target cells, predominantly erythroid cells, but also immune and hepatic cells, via a highly specialised method of receptor-mediated endocytosis (**Figure 1**). Patches of cell-surface membrane bearing receptor–ligand complexes invaginate to create clathrin-coated endosomes as distinct transferrin binds to transferrin receptor 1 (TfR1) at the plasma membrane (siderosomes) [16]. A ferrireductase reduces Fe3+ to Fe2+, which is subsequently transferred to the cytoplasm by DMT1, while TfR1 is recycled to the cell membrane and transferrin is lost. Mitoferrin, a mitochondrial iron importer, is important in providing iron to ferrochelatase for insertion into protoporphyrin IX and to produce haem (the penultimate step of mitochondrial haem production) within the erythroblast (**Figure 1**). There are some indications that iron might be transported straight from the siderosomes to the mitochondria in growing erythroid cells. Finally, haem exporters transport haem from mitochondria to cytosol and eliminate excess haem from erythroid cells (**Figure 1**) [16].

#### **2.3 Iron storage**

As senescent erythrocytes are phagocytosed by RES macrophages, haemoglobin iron turnover is high. Haem is metabolised by haem oxygenase within the phagocytic vesicles, and the liberated Fe2+ is transported to the cytoplasm by NRAMP1 (natural resistance-associated macrophage protein-1), a transport protein related to DMT1 (**Figure 1**). Macrophages may also acquire iron from bacteria and apoptotic cells, as well as from plasma via the actions of DMT1 and TfR1 (**Figure 1**) [17]. Iron may be stored in the cells in two ways: ferritin in the cytosol and haemosiderin in the lysosomes when ferritin is broken down. Haemosiderin is found in just a small percentage of normal human iron reserves, primarily in macrophages, but it rises substantially when the body is overloaded with iron. Iron storage in macrophages is also safe since it does not cause oxidative damage. Ferroportin 1, the same iron-export protein found in the duodenal enterocyte, and caeruloplasmin2 are largely responsible for iron export from macrophages to transferrin (**Figure 1**) [18]. Macrophage iron recycling provides the majority of the iron necessary for the daily synthesis of 300 billion red blood cells (20–30 mg). While a result, internal iron turnover is required to satisfy the bone marrow needs for erythropoiesis, as daily absorption (1–2 mg) only balances daily loss. 1–3 The liver is the other major iron storage organ, and the production of free radicals and lipid peroxidation products in iron-overload conditions can lead to hepatic tissue damage, cirrhosis, and hepatocellular cancer [19]. TfR1 and TfR2 mediate the liver's absorption of transferrin-bound iron from plasma (**Figure 1**), however, it can also get iron from non-transferrin-bound iron (through a carrier-mediated mechanism similar to DMT1), ferritin, haemoglobine–haptoglobin complexes, and haeme–haemopexin complexes. Ferroportin 1 is thought to be the sole protein that mediates the export of iron from hepatocytes, which is then oxidised by caeruloplasmin and attached to transferrin2 (**Figure 1**). Heart failure is the primary cause of death in individuals with untreated hereditary haemochromatosis or transfusionassociated iron overload, thus iron storage in cardiomyocytes is of significant interest. Excess iron in cardiac cells can cause oxidative stress and impair myocardial function owing to DNA damage caused by hydrogen peroxide via the Fenton reaction [20].

#### **2.4 Regulation of iron homoeostasis**

Body iron reserves, hypoxia, inflammation and erythropoiesis rate all influence iron absorption by duodenal enterocytes. The crypt programming model and the hepcidin model are two regulatory models that have been presented as potential contributors to iron absorption control [21].

Enterocytes in the crypts of the duodenum take up iron from the plasma via TfR1 and TfR2, according to the crypt programming hypothesis. The interaction of cytosolic iron regulatory proteins (IRPs) 1 and 2 with iron-responsive elements is controlled by intracellular iron content (IREs). IRP1 binds to the IREs of TfR1, DMT1, and ferroportin 1 mRNA in the absence of iron, stabilising the transcript, allowing translation to occur and the proteins to be synthesised. As a result, increased IRP-binding activity indicates low body iron reserves, which leads to overexpression of these proteins in the duodenum, boosting dietary iron absorption. When IRPs attach to ferritin mRNA's IREs, the transcript's translation is interrupted and synthesis is halted. As a result, ferritin concentrations are inversely controlled, increasing in iron-rich states and decreasing in iron-deficient conditions [22].

The hepcidin model proposes that hepcidin is produced mainly by hepatocytes in response to the iron content of the blood. Then, hepcidin is secreted into the bloodstream and interacts with villous enterocytes to regulate the rate of iron absorption by controlling the expression of ferroportin 1 at their basolateral membranes. The binding of hepcidin to ferroportin 1 initially causes Janus kinase 2-mediated tyrosine phosphorylation of the cytosolic loop of the carrier protein, phosphorylated ferroportin 1 is then internalised, dephosphorylated, ubiquitinated and ultimately degraded in the late endosome/lysosome compartment. Ferroportin 1 molecules, present in macrophages and liver, also targets for hepcidin [23].

The sensing process most likely includes local iron-induced synthesis of bone morphogenic proteins (BMPs) such as BMP6 within normal iron concentration limits. BMP6 interacts with hepatocyte cell surface BMP receptors (BMPRs) I and II, as well as the BMP coreceptor, haemojuvelin (HJV), triggering an intracellular signal by phosphorylation of small mothers against decapentaplegic (Smad) proteins. Before translocating to the nucleus and triggering hepcidin expression14, phosphorylated Smad1, Smad5 and Smad8 form a complex with the shared mediator Smad4 (**Figure 2**). The soluble form of HJV (sHJV), whose release (HJV shedding) is prevented by rising extracellular iron concentrations, is thought to compete with its membrane-anchored counterpart for BMPR binding, resulting in iron-sensitive hepcidin expression16 (**Figure 2**). Other mediators and modulators, including Smad6 and Smad7, may be stimulated by iron, and these mediators and modulators appear to dampen the signal for hepcidin activation (**Figure 2**) [24].

#### **2.5 Effects of inflammation on iron homoeostasis and erythropoiesis**

Cancer, rheumatoid arthritis, inflammatory bowel disease, congestive heart failure, sepsis and chronic renal failure are all known to induce persistent inflammation. This anaemia might be caused by the underlying process activating the immune system, as well as immunological and inflammatory cytokines such as tumour necrosis factor alpha (TNFa), interferon-gamma (IFNg), interleukins (IL) 1, 6, 8, and 10. Several pathophysiological processes (cytokines) may be implicated in anaemia of chronic disease (ACD) (**Figure 3**) [25]:


### **3. Defects of iron metabolism**

#### **3.1 Iron deficiency**

In the human body, there is a balance between iron absorption, iron transit and iron storage under physiological circumstances. ID and iron deficiency anaemia (IDA) can be caused by a combination of three risk factors: higher iron needs, restricted external supply and increased blood loss [27]. There are two types of ID: absolute and functional. Iron reserves are reduced in absolute ID; in functional iron deficiency (FID), iron stores are full but cannot be mobilised as quickly as needed from the RES macrophages to the bone marrow. Diagnostic tests with values are given in **Table 1**.


#### **Table 1.**

*Depicting tests required for determination of iron metabolism anaemia.*

#### *3.1.1 Iron deficiency anaemia*

Patients with low Hb (13 g/dl for males and 12 g/dl for women), TSAT (20%) and ferritin (30 ng/ml) concentrations but no indications of inflammation should be evaluated to have IDA. Instead of 'mean corpuscular volume (MCV)', the MCH has emerged as the most significant marker for red cells for identifying ID in RBCs, which are circulating (**Figure 1**). MCV is a generally available and reliable measurement, although it is a late indication in individuals who are not bleeding actively [28]. When MCV is low, thalassaemia must be considered a differential diagnosis. When there is a concurrent folate deficiency or vitamin B12, reticulocytosis post-bleeding, early response to oral iron therapy, alcohol use, or moderate myelodysplasia, individuals may present with IDA but no microcytosis. Human serum contains a shortened, 'soluble version of the transferrin receptor (sTfR)', whose concentration is proportional to the total number of cell surface transferrin receptors [29]. Although the amount is not defined and depends on which reagent kit is used, normal median values are 1.2–3.0 mg/l. Even during chronic illness anaemia, increased sTfR values suggest ID. Elevated erythropoietic activity without ID, during reticulocytic crises, and in congenital dyserythropoietic anaemias are all examples of increased sTfR levels. Lower sTfR levels, on the other hand, might indicate a reduction in the number of erythroid progenitors. Despite the fact that sTfR levels in simple IDA are generally high or extremely high, they are not usually necessary for diagnosis [30].

#### *3.1.2 Anaemia of chronic disease*

The following should be present in patients with chronic disease anaemia (ACD), also known as anaemia of inflammation: Hb concentration of 13 g/dl for men and 12 g/dl for women; a low TSAT (20%) but normal or increased serum ferritin concentration (>100 ng/ml) or low serum ferritin concentration (30e100 ng/ml) Evidence of chronic inflammation (e.g. elevated CRP); and a s ACD, like FID, is common in people with inflammatory illness but no visible blood loss (e.g. rheumatoid arthritis, renal failure or chronic hepatitis) [31].

#### **3.2 Iron overload**

Levels of Hepcidin are excessively lower-degree of overload of iron in idiopathic iron overload illness and primary haemochromatosis. This is due to mutations in the genes that code for 'HFE (haemochromatosis type 1)', 'haemojuvelin (HJV; juvenile haemochromatosis 2a)', and 'transferrin receptor 2 (TfR2; haemochromatosis type 3)'; these mutations cause hepcidin synthesis to be dysregulated [32]. The only exceptions are mutations that disrupt hepcidin or ferroportin (juvenile haemochromatosis 2b) (haemochromatosis type 4). Low plasma hepcidin causes high ferroportin levels, allowing for greater iron absorption, hepatic iron overload and low iron levels in macrophages. In addition, non-transferrin bound iron emerges as transferrin gets saturated in iron-overload situations. A portion of this labile plasma iron is extremely reactive, resulting in the production of free radicals. Despite the fact that the HFE gene has at least 32 mutations, the most prevalent form of haemochromatosis type 1 is caused by the missense Cys282Tyr mutation. Haemochromatosis type 1 is a disease with a wide range of penetrance and heterogeneity, although the Cys282Tyr mutation is found in the great majority of people with the disorder. Because the Cys282Tyr mutant HFE protein is unable to bind b2 microglobulin, it does not reach the cell membrane, resulting in a misfolded, non-functional protein. Iron overload can be caused by mutations in the ferroportin gene (haemochromatosis type 4) that result in the loss of iron-export capacity, hyperferritinaemia with no increase in transferrin saturation, and macrophage iron overload, or a loss of hepcidin-binding activity, which has been linked to iron overload. Plasma hepcidin levels rise in cases of secondary iron overload-induced by persistent transfusion treatment (e.g. severe thalassemia, aplastic anaemia, etc.), prompting ferroportin breakdown. Increased amounts of diferric transferrin, which are elevated in iron overload, promote TfR2 expression at the hepatocyte membrane. When diferric transferrin binds to TfR2, HJV cleavage by furin is blocked, inhibiting the release of soluble HJV and resulting in enhanced cell-surface HJV-mediated response to BMPs and higher hepcidin levels. Iron absorption from the stomach is restricted, macrophage export is inhibited, and iron storage is increased when ferroportin levels are low [33].

#### **3.3 Assessment of defective iron metabolism**

### *3.3.1 Laboratory assessment of ID*

Measurements indicating iron depletion in the body and measurements indicating iron-deficient red cell production are the two types of laboratory tests used to investigate ID (**Table 1**). The right mix of these blood tests will aid in determining the precise diagnosis of anaemia and ID status (**Figure 1**).

### *3.3.2 Assessment of iron overload*

The first step in diagnosing iron overload is to suspect it (e.g. dark skin, fatigue, arthralgia, cardiomyopathy, hepatomegaly, endocrine disorder, etc). However, aberrant TSAT (>45 per cent) and/or elevated ferritin in serum (>200 ng/ml in women, >300 ng/ml in males) are commonly discovered. In practice, normal transferrin saturation can be used to rule out the possibility of iron overload. The sole exception is the occurrence of an inflammatory state, which might disguise an increase in TSAT, which is why CRP and transferrin saturation should be checked jointly.

*Abnormal Iron Metabolism and Its Effect on Dentistry DOI: http://dx.doi.org/10.5772/intechopen.104502*

In non-iron-overload circumstances, such as significant cytolysis (eg. acute hepatitis), which raises plasma serum iron and/or hepatic failure, reduces plasma transferrin concentrations, elevated TSAT can be detected. Other causes of hyperferritinaemia should be checked out in the presence of elevated ferritin in serum but not increased TSAT (eg. cell necrosis, alcohol, inflammation, metabolic disorder, etc). The clinical context, as well as testing Hb (to rule out chronic inflammatory anaemia), transaminases, cancer and prothrombin index, can readily remove any difficulties in interpreting TSAT readings (to exclude hepatic disease) [34].

The second diagnostic step, particularly in Caucasian individuals, is to rule out HFE mutations in gene. Because further mutations in HFE are exceedingly rare, the HFE genotype is frequently regarded as 'wild type' in clinical practice, once the presence of the two most prevalent (Cys282Tyr and His63Gly) mutations has been ruled out. Nonetheless, the potential of a family problem should be addressed at all times: a dominant disorder is usually indicative of ferroportin disease [35].

Before beginning costly and time-consuming searches for mutations in additional genes, the third diagnostic step is to establish increased total body iron. The exact molecular diagnosis, which needs evidence of the nucleotide mutation at the DNA level, is the fourth stage. However, the efficacy of molecular diagnostics is frequently questioned because it is costly, time-demanding and, in certain situations, unable to produce a precise diagnosis [36].

#### **4. Iron metabolism and the oral health**

#### **4.1 Iron deficiency anaemia**

The most prevalent kind of anaemia is iron deficiency anaemia (IDA), which affects more women than males. Due to persistent blood loss associated with heavy menstrual flow, it is estimated that 20% of women of reproductive age in the United States are iron deficient. Furthermore, 2% of adult males are iron deficient due to persistent blood loss caused by gastrointestinal illnesses including peptic ulcer, diverticulosis, or cancer [37].

#### *4.1.1 Symptoms*

Atrophic glossitis (AG), extensive oral mucosal atrophy and pain or burning feeling of the oral mucosa are some of the oral symptoms and indicators. However, it is yet unknown if IDA patients may experience distinct oral signs and, if so, what percentage of IDA patients experience these oral manifestations. Burning sensation of the oral mucosa (76.0 per cent), lingual varicosity (56.0 per cent), dry mouth (49.3%), OLP (33.3 per cent), AG (26.7 per cent), RAU (25.3 per cent), numbness of the oral mucosa (21.3 per cent) and taste dysfunction (12.0 per cent) were the most commonly manifested oral manifestations. IDA patients had considerably greater rates of all oral symptoms, such as oral mucosa burning, lingual varicosity, dry mouth, oral mucosa numbness, and taste impairment than healthy controls.

#### *4.1.2 Pathophysiology*

Anaemia sufferers have low haemoglobin levels, which means they do not get enough oxygen to their mouth mucosa, causing it to atrophy. Iron deficiency can induce oral mucosa atrophy because iron is required for proper oral epithelial cell activity, and in an iron deficiency condition, oral epithelial cells turn over more quickly, resulting in an atrophic or immature mucosa. The health of the oral epithelium is linked to iron and vitamin B12.

In BMS patients, long-term dry mouth and iron or vitamin B12 deficiency may produce at least partial atrophy of the tongue epithelium, however, the change is so mild that clinical visual examination cannot detect it. As a result, spicy chemicals in saliva might readily permeate past the atrophic epithelium into the subepithelial connective tissue of the tongue mucosa, irritate free sensory nerve endings, and cause tongue burning and numbness. A minor sign of BMS was loss or malfunction of taste. Because the taste cells in taste buds can only sense dissolved compounds, the chemical components should be dissolved in saliva.

The majority of BMS patients were found to have xerostomia. In BMS patients, decreased saliva output leads to a loss or malfunction of taste. Oral candidiasis, vitamin B12 insufficiency, iron deficiency and medicine have all been linked to taste loss or malfunction. Femiano et al. have looked into the causes of taste disturbance in BMS patients. Of the 142 BMS patients, 61 had a documented history of drug use that interfered with taste perception, 35 had pathologies or a past history of drug use that were known to impact the gustatory system, and the other 46 had no related disease or regular drug use [38].

Varicosities are abnormally dilated, and convoluted veins are observed on the ventral surface of the tongue in elderly people due to a decrease in connective tissue tone that supports the veins. Furthermore, xerostomia is a prevalent issue that affects 25% of the elderly population. Xerostomia can be caused by a variety of developmental, iatrogenic, systemic and local causes. Older individuals, on the other hand, are more likely to develop xerostomia, as a result of pharmaceuticals, as they are more likely to use drugs that induce xerostomia to treat their systemic or psychotic diseases. The average age of 399 BMS patients in Wang Y et al's research was 59.7 years. As a result, it is not unexpected that 92.5 per cent of 399 BMS patients had lingual varicosity and 75.7 per cent had dry mouth. Oral candidiasis is more common in persons with xerostomia because normal and adequate saliva can offer cleaning and antibacterial action. We believe that the candidiasis on the tongue surfaces of BMS patients is attributable, at least in part, to the high prevalence of dry mouth (75.7%).

#### *4.1.3 Management*

#### *4.1.3.1 Oral iron*

In most therapeutic situations, oral iron supplementation is sufficient. In the absence of inflammation or severe continuous blood loss, oral iron, usually in the form of ferrous salts, can be used to treat anaemia if large dosages are tolerated. Although traditional knowledge holds that up to 200 mg of elemental iron per day is necessary to treat IDA, this is erroneous and lesser amounts can be effective as well.

Early research suggested that taking iron with vitamin C might help with iron absorption because more ferrous iron is kept in the solution. However, findings suggest that co-administration of these drugs might cause serious toxicity in the gastrointestinal tract. Furthermore, while taking oral iron away from meals is often suggested to increase absorption, it also increases gastric intolerance, which reduces compliance. Furthermore, some antibiotics (primarily quinolones, doxycycline, tetracycline, chloramphenicol, or penicillamine), proton pump inhibitors, and anti-acid

#### *Abnormal Iron Metabolism and Its Effect on Dentistry DOI: http://dx.doi.org/10.5772/intechopen.104502*

medication (aluminium, bicarbonate, zinc, or magnesium salts), levodopa, levothyroxine, cholestyramine, phytates (high-fibre diets), soy products, ibandronate, etc.

Non-absorbed iron salts, on the other hand, can produce a variety of highly reactive oxygen species, such as hypochlorous acid, superoxides and peroxides, which can cause digestive intolerance, resulting in nausea, flatulence, abdominal pain, diarrhoea or constipation and black or tarry stools, as well as relapsed inflammatory bowel disease. As a result, smaller iron salt dosages (e.g. 50–100 mg elemental iron) should be advised. The Ganzoni method may be used to determine the total iron deficiency (TID): TID (mg) 14 weight (kg) 3 (ideal Hb e actual Hb) (g/dl) 3 0.24 + depot iron (500 mg). An individual, weighing 70 kg, with a haemoglobin level of 9 g/dl would have a body iron shortfall of around 1400 mg, according to this calculation.

#### *4.1.3.2 Parenteral iron*

Parenteral iron is traditionally used to treat intolerance, contraindications, or an insufficient response to oral iron. However, in circumstances when there is a limited time until surgery, severe anaemia, especially if it is accompanied by considerable continuous bleeding or the use of erythropoiesis-stimulating drugs, parenteral iron is now an effective therapy. Because they provide various benefits over oral supplements, modern intravenous iron formulations have emerged as safe and effective options for anaemia therapy. In normal persons, intravenous iron delivery allows for a fivefold erythropoietic response to substantial blood loss anaemia,19 Hb begins to rise after a few days, the percentage of responsive patients increases, and iron reserves are replenished. Increasing iron reserves is beneficial, especially for patients using erythropoiesis-stimulating drugs. In clinical practice, iron gluconate, iron sucrose, high molecular weight iron dextran (HMWID), low molecular weight iron dextran (LMWID), ferric carboxymaltose, iron isomaltoside 1000 and Ferumoxytol are the most commonly used products.

#### *4.1.4 Changing microflora in patients with ida and its corelation with infective endocarditis*

The link between oral microbiota and IE (infectious endocarditis) has long been known. Infectious endocarditis is caused by opportunistic infections in normal oral flora entering the circulation through everyday mouth washing or invasive dental treatments. In vitro iron deficiency causes a dramatic change in the oral microbiota community, with higher proportions of taxa linked to infective endocarditis. Iron deficiency anaemia is utilised as an in vivo model to evaluate the association between insufficient iron availability, oral microbiota, and the risk of IE, as well as to perform population amplification research. In a research by Xi R et al., 24 patients with primary iron deficiency anaemia (IDA) from the haematology department of West China Hospital, Sichuan University, and 24 healthy controls were included from 2015.6 to 2016.6. The dental plaque microbiota of 24 IDA (iron-deficiency anaemia) patients and 24 healthy controls were compared using high-throughput sequencing. Internal diversity in the oral flora is reduced as a result of iron shortage. Corynebacterium, Neisseria, Cardiobacterium, Capnocytophaga and Aggregatibacter had considerably greater proportions in controls, whereas Lactococcus, Enterococcus, Lactobacillus, Pseudomonas and Moraxella had significantly larger proportions in the IDA group (P 0.05). Lactococcus, Enterococcus, Pseudomonas and Moraxella relative abundances were substantially inversely linked with serum ferritin concentrations (P 0.05). In vivo iron shortage altered the organisation of the oral microbiome population. When compared

to healthy controls, people with IDA had lower total bacterial diversity and different taxonomic makeup. The IDA group had greater proportions of the genera Lactococcus, Enterococcus, Pseudomonas and Moraxella, whose abundance was likewise statistically and adversely linked with serum ferritin levels. Because the IDA group has a high rate of penicillin resistance, the typical use of preventive penicillin may be ineffective. The findings of a disproportionate oral microbiota suggest that more targeted antibiotic usage with various groups may be required before dangerous oral surgeries.

### **4.2 Iron overload**

Hemochromatosis is the abnormal accumulation of iron in parenchymal organs, leading to organ toxicity. It is the most common inherited liver disease in whites and the most common autosomal recessive genetic disorder. Genetic haemochromatosis (GH), which is related to the HFE gene p.Cys282Tyr mutation, is the most common form of inherited iron overload disease in European population descendants.

### *4.2.1 Symptoms*

The classic tetrad of manifestations resulting from hemochromatosis consists of: (1) cirrhosis, (2) diabetes mellitus, (3) hyperpigmentation of the skin and teeth, and (4) cardiac failure. Clinical consequences also include hepatocellular carcinoma, impotence and arthritis (**Figures 4** and **5**) [9].

Symptoms can vary from burning mouth syndrome to bald and inflamed tongue [9].

**Figure 4.** *Tongue anomaly of iron deficiency anaemia.*

**Figure 5.** *Balding of tongue seen due to iron deficiency anaemia.*

#### *4.2.2 Pathophysiology*

Periodontitis is linked to an inflammatory response triggered by changes in the subgingival biofilm. Inflammation causes iron sequestration inside macrophages in healthy people, depriving bacteria of iron. Iron bioavailability in biological fluids, particularly those of the oral cavity, is enhanced in GH patients with excessively high TSAT, resulting in an increased risk of severe periodontitis. The existence of iron deposits in oral tissues of haemochromatosis patients has also been documented in the literature (**Figure 6**). The majority of people with haemochromatosis are now asymptomatic, and the skin and mucosal colouration caused by iron deposits have improved dramatically. The occurrence of asymptomatic iron deposits in oral tissues, however, cannot be ruled out [10, 11].

Iron is connected with transferrin in plasma, which increases its bioavailability for cells. The ratio between the total number of iron-binding sites on patient plasma transferrin and the number of binding sites occupied by iron is known as transferrin saturation (TSAT). TSAT is normally seen in the range of 20% to 45 per cent. Hepcidin regulates systemic iron metabolism, and its expression level is tuned to TSAT to regulate plasma iron levels. Hepcidin insufficiency is a symptom of GH, which is caused by a change in the HFE-linked transduction signalling pathway. TSAT levels rise as a result of the iron outflow from macrophages and enterocytes. Non-transferrin-bound iron (NTBI), an aberrant biochemical type of iron, arises in the plasma when TSAT surpasses 45 per cent. The liver and heart are particularly vulnerable to NTBI, which explains why the typical type of GH causes hepatic cirrhosis and diabetes. However, in the absence of cirrhosis or diabetes, the majority of GH patients remain asymptomatic or have chronic tiredness, abnormal serum transaminase levels, rheumatism, and osteoporosis. Cells manufacture ferritin to store excess

**Figure 6.** *Staining of teeth seen due to iron deficiency anaemia.*

iron in order to avoid iron toxicity. As a result, the tissue iron reserves are reflected in plasma ferritin levels. The standard treatment is phlebotomy therapy, which is used to take out excess iron and then prevent it from being reconstituted. The gold standard for both initial treatment and maintenance therapy, according to the leading international standards, is serum ferritin levels of less than 50 g/L [13].

#### *4.2.3 Management*

Iron depletion would lessen or eliminate the risk of iron-mediated tissue harm, according to the earlier reasoning for blood removal in all patients with haemochromatosis. This may help to avoid or lessen the severity of some haemochromatosis problems after iron deficiency. Dyspnoea, pigmentation, weariness, arthralgia, or hepatomegaly may be reduced, and diabetes mellitus management and left ventricular diastolic function may be improved. The progression of hepatic cirrhosis, as well as the increased risk of primary liver cancer, hyperthyroidism and hypothyroidism, are largely unaffected.

Standard therapy for most patients with haemochromatosis and iron overload is weekly blood removal to bring ferritin levels into the low reference range (20–50 ng/ ml), followed by a life-long maintenance phlebotomy schedule to maintain ferritin levels at around 50 ng/ml, for preventing or treating iron overload. The number of units to be removed can be calculated using the following formula: 1 ng/ml ferritin corresponds to nearly 8 mg mobilisable iron in the absence of hepatic necrosis

#### *Abnormal Iron Metabolism and Its Effect on Dentistry DOI: http://dx.doi.org/10.5772/intechopen.104502*

or another source of inflammation that causes hyperferritinaemia, and a 500 ml blood unit contains approximately 200 mg iron. To achieve iron depletion, a patient with serum ferritin of 1000 ng/ml will likely require the removal of 40 units of blood. Traditional phlebotomy or erythrocytapheresis can be used to remove blood. Traditional phlebotomy (250–500 mL once or twice weekly during the initial phase, depending on patient's characteristics and level of iron overload, followed by 500 mL every 2–4 months for the rest of one's life) is effective for iron depletion, but it necessitates normal erythropoiesis and frequent visits to a healthcare facility, and some patients report intolerance. Blood taken for therapeutic phlebotomy at blood donation facilities can be used to supplement the blood supply for transfusion, according to new US Food and Drug Administration rules (Title 21, Code of Federal Regulations, Section 640.120). (21 CFR 640.120). Isovolaemic, large-volume erythrocytapheresis, on the other hand, removes more blood erythrocytes each session than phlebotomy while leaving plasma proteins, coagulation factors, and platelets alone. As a result, therapeutic erythrocytapheresis is a quick and safe procedure that may be recommended in the early stages of treatment for individuals with significant iron excess. Although a single therapeutic erythrocytapheresis session is more expensive, the overall expenditures to cause iron depletion are comparable to or less expensive than therapeutic phlebotomy; yet, the treatment is only available in limited quantities (special apparatus and facilities, trained personnel, etc). Both treatments, however, have comparable side effects: transitory hypovolaemia; weariness (Hb levels should not go below 11 g/dl); enhanced iron absorption; citrate response (erythrocytapheresis alone); or iron insufficiency if proper monitoring is not performed. Iron chelation therapy, on the other hand, is seldom optimal for patients with haemochromatosis, unless they are unable to undertake phlebotomy therapy due to expense, probable toxicity and a lack of proof of benefits. Finally, while dietary restrictions (e.g. low meat consumption, abstinence from alcohol, restricted use of vitamin and mineral supplements, etc.) and medications to reduce iron absorption (e.g. proton pump inhibitors) appear to be reasonable options for patients with haemochromatosis, they have yet to be evaluated in prospective randomised clinical trials [14].

#### *4.2.4 Iron chelation therapy*

In patients with acquired iron overload (e.g. anaemia dependent on transfusion), iron-excess management and management of toxicity due to excess iron with chelation have been shown to lower iron burden and increase survival. Patients with serial serum ferritin levels more than 1000 ng/ml and a total infused red blood cell volume of 120 ml/kg of body weight or higher should be treated with chelation treatment, according to recent consensus recommendations. During chelation therapy, serum ferritin levels should be checked every three months to determine that the medication is effectively lowering iron levels. Deferasirox is cost-effective when compared to standard parenteral iron chelation therapy with deferoxamine, according to cost analyses conducted in the United Kingdom and the United States. This is primarily due to the quality-of-life benefits derived from the simpler and more convenient mode of oral administration. The first results from a phase I/II investigation of deferasirox in HFE-haemochromatosis show that a dosage of 5–10 mg/kg/day is sufficient to decrease iron burden, and a randomised trial comparing deferasirox to phlebotomy is now underway [32].

### **5. Iron metabolism and its effect on caries, microhardness of tooth and discoloration**

Although research on iron salts compounds and iron ions support the cariostatic concept, it is difficult to make definitive statements about iron loss owing to a range of chemicals and additives. In the context of a cariogenic diet, however, it appears that specific drops in iron content have a static effect on caries. In light of the current data, it is likely reasonable to state that if a kid consumes carbohydrates that are utilised by cariogenic bacteria, the cariostatic impact might be calculated based on iron drop intake (especially the form of ferrous sulfate. Ferrous Sulphate affects the most as proven in the literature) [33].

In a case–control study by Schroth et al. which aimed to contrast ferritin and haemoglobin levels between preschoolers with S-ECC and caries-free controls, it was concluded that children with S-ECC (severe early childhood caries) appear to be at significantly greater odds of having low ferritin status compared with caries-free children. Children with S-ECC appear to have significantly lower haemoglobin levels and appear to be at significantly greater odds for iron deficiency when compared with caries-free controls.

In the realm of microhardness, the presence of iron in combination with sucrose has resulted in a decrease in the microhardness changes of cow and human enamel. Furthermore, in both in vitro and in vivo conditions, adding iron to acidic liquids reduces demineralization. There is still debate over the mechanism of action of such an ion and its different forms, and this is a fascinating study subject.

Consumption of iron-rich foods (eggs, vegetables, etc.) tends to promote the bacterial growth that produces colouration which is black in the teeth. It has been shown that children with black pigmentations have more calcium and phosphate in their saliva, which can boost the saliva's buffering qualities and lead to a reduction in the occurrence and prevention rate of decay in the presence of pigmentation. However, the relationship between pigmentation, food, oral flora decay and has yet to be found. The combination of iron and sulphide ions produced by bacteria activity is mainly responsible for the iron drop's colour. To justify no indication of colour change in all consumers, the colour change varies with varied iron drop consumption, which might be connected to the total quantity of iron accessible in each drop, the acidity and drops' capacity to etch the surface of the tooth, any bacterial flora, individual's diet and so on [39].

#### **6. Conclusion**

The study of microbial iron metabolism is gaining popularity. Initial research on the subject revealed the many ways in which bacteria get iron, began to unravel the crucial function of iron in bacterial metabolism and revealed the means and demand for precise iron absorption management. Iron's role in bacterial pathogenesis has been well documented, and it is currently taken into account in all investigations of prokaryotic pathogens. Basic investigations using *E. coli* and its relatives have given way to studies of less known, and more difficult to grow, organisms, although still incomplete and giving unexpected discoveries, such as the discovery of glucosylated derivatives of enterobactin. Biogenesis research in magnetotactic bacteria has the potential to identify pathways that govern biomineralisation and give insight into organelle development. The potential biotechnology implications of dissimilatory iron reduction research are

*Abnormal Iron Metabolism and Its Effect on Dentistry DOI: http://dx.doi.org/10.5772/intechopen.104502*

also intriguing. Because of the extensive and essential role played by environmental interactions between bacteria and iron, geologists, ecologists, environmental and chemical engineers, and physicists, among other professions, have entered the topic. There is a good chance that numerous exciting new discoveries will be made.

## **Author details**

Chinmayee Dahihandekar\* and Sweta Kale Pisulkar Department of Prosthodontics, Sharad Pawar Dental College and Hospital, Maharashtra, India

\*Address all correspondence to: chinmayeead@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### **Chapter 6**

## Ferroptosis: Can Iron Be the Downfall of a Cell?

*Asuman Akkaya Fırat*

#### **Abstract**

Ferroptosis is one of the forms of programmed cell death. Besides being a necessary micronutrient, iron is the key element that initiates ferroptosis in the cell. Intracellular unstable iron accumulation increases the amount of intracellular ROS, especially by the peroxidation of unsaturated membrane phospholipids. Insufficient antioxidant capacity and decreased glutathione levels play an important role in this process. The research reveals that an imbalance between unoxidized polyunsaturated fatty acids (PUFAs) and oxidized PUFAs, particularly oxidized arachidonic acid, accelerates ferroptosis. These oxidative reactions change the permeability of lysosomal and cellular membranes and cell death occurs. Iron chelators, lipophilic antioxidants, and specific inhibitors prevent ferroptosis. In addition to being accepted as a physiological process, it seems to be associated with tissue reperfusion damage, ischemic, neurodegenerative diseases, hematological and nephrological disorders. Ferroptosis is also being explored as a treatment option where it may offer a treatment option for some types of cancer. In this section, the brief history of ferroptosis, its morphological, molecular, and pathophysiological features are mentioned. Ferroptosis seems to be a rich field of research as a treatment option for many diseases in the future.

**Keywords:** ferroptosis, iron, RCD (regulated cell death), ROS (reactive oxygen species), lipid peroxidation (LP)

#### **1. Introduction**

Iron is an essential micronutrient for all living cells. Microorganisms, plant, animal, and human cells need iron to sustain their vital reactions. However, iron overload can cause various metabolic problems and even cell death. Ferroptosis, which has been revealed in the past years, is a form of regulated cell death that develops depending on the increase in the iron load in the cell [1, 2].

Recently, many new cell death modalities have been described. All cell death is considered primarily "regulated cell death" (RCD) and "accidental cell death" (ACD). The accidental cell death occurs in the cell exposed to chemical and physical attacks, independent of genetic coding and molecular pathways. Accidental cell death is a biologically uncontrolled process. Whereas regulated cell death requires signaling cascades and biologically effector molecules. RCD includes apoptosis, necroptosis, autophagy, ferroptosis, pyroptosis, entotic cell death, netotic cell death, parthanatos, lysosome-dependent cell death, alkaliptosis and oxeiptosis [1].

RDC was first observed in the dying cells of frogs by Karl Vogt in 1842 [1]. Kerr coined the term "apoptosis" for the first time in 1972. Kerr et al. defined apoptosis as a form of programmed cell death (PCD) with morphological changes that differ from necrosis [2]. A new milestone was the identification of CED9 (also known as BCL2 in mammalian cells) and CED4 (also known as apoptotic peptidase activating factor 1 [APAF1] in mammalian cells) from Caenorhabditis elegans development studies in the 1990s [1, 3–5]. RCD is also known as PCD when it occurs in physiological conditions [6]. Apoptosis is considered one of the main reference forms when examining cell death models. Thus, studies on apoptosis accelerated and it was revealed that it can develop in two different ways as intrinsic and extrinsic apoptosis. In recent years, many research results have been revealed about other sub-titles of RCD. The current classification system of cell death has been updated by the Nomenclature Committee on Cell Death (NCCD), which formulates guidelines for the definition and interpretation of all aspects of cell death since 2005 [7].

Ferroptosis is a new type of programmed cell death. In 2003, Dolma et al. discovered the molecule erastin (ST), which has a selective lethal effect on cancer cells that express the RAS family of small GTPases (HRAS, NRAS, and KRAS) protein. The pattern of cell death induced by erestin was different from the previous ones. This new form of cell death did not show any nuclear morphological changes, DNA fragmentation, or caspase activation. In addition, this process was irreversible with caspase inhibitors [8]. Yang and Yagoda found the RSL3 (Ras select and lethal 3) component that inhibits this cellular death pattern and revealed that this cell death process can be stopped by iron chelators [8–10]. Erastin and RSL3 treatment do not induce morphological changes consistent with apoptosis, such as cleavage of ADP-ribose polymerase (PARP). The mechanism of cell death induced by erastin and RSL3 is not attenuated by deletion of the intrinsic apoptotic effectors BCL-2-associated X protein (BAX) and a small molecule inhibitor of BCL-2 antagonist/killer 1 (BAK). These differences distinguish the newly described cell death mechanism from apoptosis, autophagy, and necroptosis. Furthermore, neither mitochondrial ROS production nor Ca+2 influx is required for cell death in ferroptosis to occur. Erastin has also been found to cause mitochondrial dysfunction by affecting voltage-dependent anion channels (VDAC) [10]. The term ferroptosis was first used by Dixon et al. For cell death in cancer cells with RAS mutations in 2012. This newly recognized form of cell death can be initiated by iron accumulation and prevented by iron-binding chelators. That's why it's called ferroptosis [8–11].

#### **2. Morphological features of ferroptosis**

Ferroptosis is characterized as a cell death model defined as morphologically, reduced mitochondrial volume, decreased or completely absent mitochondrial cristae, increased bilayer membrane density, while the cell membrane is intact, the nucleus remains normal in size, and there is no increase in chromatin density [10, 11]. On electron microscopy, it looks similar to the typical dysmorphic mitochondrial appearance caused by Erastin treatment [12]. Biochemically, intracellular glutathione (GSH) depletion, decreased activity of glutathione peroxidase 4 (GPX4) enzyme, inability to metabolize lipid peroxides, and accumulation of large amounts of ROS (Reactive Oxygen Space) due to iron initiate a lethal process similar to Fenton's reaction and genetically regulated by many genes that have not yet been elucidated [13]. Cancer cells with highly active RAS-RAF-MEK (Receptor Tyrosine Kinases) pathways are susceptible to ferroptosis. The genetic mechanisms that regulate ferroptosis may be related to iron homeostasis and lipid peroxidation [14]. Ferroptosis shows similarities to pathways in other RDC types. Iron-dependent lipid peroxide accumulation is considered to be the basis of the ferroptosis mechanism. It is thought to be a physiological process in mammals rather than a disease or pathological process [15, 16].

### **3. Accumulation of lipid peroxidase**

Mainly phosphatidylethanolamine-OOH (PE-OOH), lipid peroxides are reduced to appropriate lipid alcohols (PE-OOH) by antioxidant reductase mechanisms in the cell under physiological conditions. The effect that will initiate ferroptosis is either by increasing lipid peroxides or by inhibiting the reduction pathway. Glutathione (GSH), the cofactor of glutathione peroxidase (GPX4), is important for the conversion of toxic lipid peroxides to nontoxic lipid alcohols. Glutathione is a tripeptide containing selenocysteine, glutamine, tryptophan. GPX4 catalyzes the following reaction:

> + → +− + <sup>2</sup> 2 glutathione lipid – hydroperoxide glutathione disulfide lipid alcohol H O. (1)

This reaction occurs in selenocysteine within the catalytic center of GPX4. During the catalytic cycle of GPX4, active selenol (∙SeH) is oxidized by peroxides to selenic acid (∙SeOH) and then reduced by glutathione (GSH) to an intermediate selenodisulfide (∙Se-SG). GPX4 is eventually reactivated by a second glutathione molecule and glutathione disulfide (GS-SG) is released [17, 18]. GPX4 contains eight neutrophilic amino acids. One of them is selenocysteine and seven cysteines. Selenium, together with cysteine, is essential for the function of GPX4. Inactivation of GPX4 is the most important factor in increasing intracellular lipid ROS and initiating ferroptosis [17–19]. Firstly, in the 1950s, Harry Eagle et al. reported that amino acids, vitamins, and other nutrients are required to protect against oxidative stress in cell culture [20, 21]. Among the molecules reported to be essential was cystine, the oxidized form of cysteine-containing thiol groups [21]. Banni et al., despite glutathione deficiency in human diploid fibroblast cell culture, were able to induce cell growth with α-tocopherol (vitamin E), a lipophilic antioxidant [22].

Compounds that stimulate ferroptosis via GPX4 are divided into four groups (i.e., erastin, RSL3, FIN56, FINO2).

#### **3.1 Erastin**

The first group includes erastin (ST). ST inhibits Xc (System Xc-cystine/glutamate antiporter) and decreases intracellular glutathione (GSH) levels. System Xc is an amino acid anti-transporter commonly found in phospholipid bilayer phospholipid membranes. It is an important part of the antioxidant system in cells. It has a heterodimer structure and consists of two subunits, SLC7A11 and SLC3A2, linked to each other by disulfide bonds. System Xc operates on a sodium-free, chlorine-dependent basis. It exchanges cysteine and glutamate in a 1/1 ratio dependent on ATP [21]. Inhibition of the Xc \_ system reduces the uptake of cystine, the oxidized form of cysteine [23]. Cysteine is used in intracellular glutathione synthesis [24]. In cells, GSH synthase and glutamate cysteine synthase synthesize GSH with glutamate, glycine, and cysteine, which is reduced from cystine in the cell as substrates [25]. Glutathione reduces the increased load of ROS and the amount of reactive nitrogen decreased cystine causes a decrease in cysteine and depletion of GSH which uses it as a cofactor, to convert lipid peroxides into suitable lipid alcohols, and an increased intracellular oxidant load [25]. Glutathione reduction and

decreased glutathione peroxidase activity increase ROS accumulation, oxidative damage, and ultimately ferroptosis [19, 24, 25]. Erastin's blocking of the Xc system and disrupting the intracellular lipid ROS balance damages all intracellular organic substances (e.g. proteins, lipids and nucleic acids), particularly lipid peroxidation initiates ferroptosis. It has been shown that erastin, a prototype compound that inhibits GPX4 via system Xc, also causes ferroptosis by affecting voltage-dependent anion channels. Early chemoproteomic studies showed that voltage-dependent mitochondrial voltage-dependent anion channels 2 and 3 (VDAC2, VDAC3) are direct targets for erastin blockade. VDCA2, purified and reconstituted as artificial liposomes, has been shown to be the target of erastin and modulates transport flow [10, 26]. However, it is accepted that erastin initiates ferroptosis mainly by blocking the X c system cystine/glutamate antitransporter [27]. Also, butionine sulfoximine (BSO), sorafenib, and artesunate induce ferroptosis by depletion of GSH [14, 28, 29]. Reagents or treatments that increase the intracellular amount of cystine/cysteine can reverse erastin-induced ferroptosis, such as β-mercaptoethanol (β-ME), transsulfuration, and processes that increase cysteine synthesis [30–32].

#### **3.2 RSL3**

RAS-selective lethal 3 (RSL3), contains an electrophilic moiety and a chloroacetamide moiety and reacts with selenocysteine in the nucleophilic eight amino acid moiety of GPX4, and the enzyme is blocked [33]. Altretamine, which is thought to have a mechanism similar to RSL3, has been defined by the FDA as an anti-cancer drug, but the mechanism of altretamine GPX4 resistance has not been clarified yet [34].

#### **3.3 FIN56**

It was named CIL 56, which causes death by ferroptosis in RAS cells while caspas 3 and 7 have no activity. The effect of CIL 56 causing cell death could only be eliminated with low doses of anti-oxidants and iron chelators. At high doses, the lethal effect was irreversible. Later found a CIL56 analog FIN56 (ferroptosis inducing 56) which preserves ferroptosis selectivity in RAS cells. The toxic small-molecule FIN56 is required for mevalonate pathway-mediated ferroptosis. FIN56 can activate its own target protein SQS besides inducing ferroptosis by decreasing the abundance of GPX4 [35]. FIN56 can cause ferroptosis in two ways, either by causing degradation of GPX4 or by reducing the amount of CoQ (i.e., an antioxidant in the cell). The enzymatic activity of acetyl-CoA carboxylase (ACC) is required for FIN56 to degrade GPX4. Therefore, the mechanism of FIN56-induced ferroptosis involves two distinct pathways in association with the mevalonate pathway and fatty acid synthesis. FIN56-mediated mevalonate pathway reduces CoQ. FIN56 binds and activates SQS, the enzyme that converts farnesyl pyrophosphate (FPP) to squalene, which ultimately reduces the level of coenzyme Q10' by reducing the FPP pool available for protein prenylation and metabolite synthesis [35].

#### **3.4 FINO2**

FINO2 (endoperoxide-containing 1,2-dioxolane) is a 1,2-dioxolan with both an endoperoxide moiety and a hydroxyl head, capable of inducing ferroptosis. Although its mechanism has not been fully resolved, it indirectly reduces the activity of GPX4. It also provides lipid peroxidation by forming oxygen-centered radicals, similar to the Fenton reaction. Ferroptosis initiated by both FIN56 and FINO2 is partially reversible by β-mercaptoethanol (β-ME) [36, 37].

### **4. Lipid peroxides and ROS increase**

Lipids are important organic molecules because they provide energy for the cell and participate in the structure of cell membranes. Oxygenation of phospholipid (PL) (e.g. PE, phosphatidylcholine, cardiolipin) facilitates ferroptosis. Lipid peroxides are produced in three different ways, each requiringan iron molecule [24, 33, 38]. 1. Lipid ROS produced non-enzymatically by the Fenton reaction with the iron molecule. 2. Lipid peroxides formed by esterification and oxidation of polyunsaturated fatty acids (PUFAs) [24, 33, 39, 40]. 3. Lipid peroxides are formed by catalyzing the iron molecule by lipid auto-oxidation [41]. Fenton reaction is an inorganic reaction that occurs commonly in nature. However, although not fully resolved, (PUFAs) are likely to be the reaction that most contributes to the ferroptosis process [12].

Kagan et al. used RSL3, known as a selective inhibitor, to induce GPX4 inhibition in mouse embryogenic fibroblast cells. RSL3 caused a marked decrease in the chemical activity of GPX4. Among the eight different forms of GPX, GPX4 is the only one that reduces PL-OOH (phospholipid hydroxyls) and PUFA (polyunsaturated fatty acid hydroxyls) in membranes. Kagan et al. screened 350 species of PLs (phospholipids) and identified oxidized AA-containing PE (acyls-arachidonoyl phosphatidyl ethanol) as a ferroptotic cell death signal. AA is a type of PUFAs that can be elongated into adrenoyl (AdA) by elongase [24]. Accumulation of oxidized AA-PE and AdA-PE causes ferroptosis in cells.

It was revealed that the molecule that induces ferroptosis is AA-OOH-PE rather than PL-OOH. The formation of AA-OOH-PE from AA in the cell requires three types of enzymes: 1. lipoxygenases (LOXs), 2. acyl-CoA synthetase long-chain family 4 (ACSL4), and 3. lysophosphatidylcholine acyltransferase 3 (LPCAT3) [11, 17, 24, 39, 40]. In this process, AA is first converted to AA-CoA by being catalyzed by ACSL4, then esterified with LPCAT3 to AA-PE, and finally to AA-OOH-PE a with AA-PE LOXs. Generally accepted views 1. Lipid autoxidation is definitely associated with ferroptosis. 2. Lipid oxidation is associated with ferroptosis rather than lipid peroxidation and is a continuation of lipid peroxidation that cannot be prevented from continuing. 3. Lipid peroxidation initiates lipid autoxidation, while lipid autooxidation causes cell death [38, 41]. In cells undergoing ferroptosis, arachidonic acid (AA) is the most affected by autoxidation. Abundant AA residues were observed in the supernatant of mouse embryo fibroblast (MEFs) with GPX4 depletion. Acyl-CoA synthetase long-chain family4 (ACSL4) and lysophosphatidylcholine acyltransferase3 (LPCAT3) encode enzymes involved in the insertion of AA into membrane phospholipids [42, 43]. ACLSs are composed of proteins expressed on the outer membrane of the endoplasmic reticulum and mitochondria. ACSLs are responsible for the formation of acyl-Cos from fatty acids. There are 5 isoforms: ACLS1, ACSL3, ACSL4, ACSL5, ACSL6 [41]. It has been reported that ACLS4 correlates with ferroptosis. ACLS4 is required for ferroptosis to occur in cells with GPX4 knockout or cells with GPX4 [39]. ACSL4 is not the only enzyme that can activate AA arachidonic acid (arachidonic acid) and ADA, but very high concentrations of AA and AdA are required for ACSL3 to activate, for example. However, AA is normally present in the cell in lower amounts than other fatty acids. Thus, ACSL4 is considered a major regulator for AA and ferroptosis [39]. The mentioned 3 enzymes (ACSL4, LPCAT3, LOX) are active in the initial phase of ferroptosis. As a result of these reactions, the amount of intracellular LOOH increases. Increased intracellular LOOH levels and low valent metals (Fe+2) initiate lipid autooxidation, which is essential for ferroptosis. Lipid autoxidation is the specific and final stage of ferroptosis. Lipid autooxidation can only be reversed by radical-trapping antioxidants (RTAs). Lipid autoxidation, rather than LOXs-directed lipid peroxidation, is considered to be the final phase

of ferroptosis that causes cell death [38]. It is accepted that intracellular RTA and LOXs levels determine the sensitivity of cells to ferroptosis. According to this assumption, sensitivity to ferroptosis is a physiological process that is affected by many variables such as cell type, physiological conditions and environmental factors [44].

#### **5. Iron and ROS**

Iron is an essential element for almost all living organisms. An adult human body contains about 3–5 g of iron. Iron in erythrocytes accounts for 80% of the total iron, and less than 20% is stored in macrophages and hepatocytes. The iron in the macrophage comes from aged red blood cells and is reused, providing 90% of the daily required iron. Approximately 1 g of iron from the diet per day is absorbed through the gastrointestinal tract as 'new iron'. Daily iron loss occurs mostly with the desquamation of epithelial cells in the skin and gastrointestinal tract. In women, menstruation and labor bleeding can cause large amounts of iron losses. The excessive increase of iron in the human body causes hemochromatosis, and less than an adequate amount causes anemia [45, 46]. Inside the cell, iron exists in two forms, Fe+2 and Fe+3. The Fe+2 form is more functional because of its ability to transfer electrons and have high solubility. The Fe+3 form is more stable chemically, so this form is suitable for storage and transportation. Fe+2 plays an important role in oxidation-reduction reactions. Fe+2 reacts with H2O2 to form hydroxyl reagent and Fe+3. Thus, the ROS load inside the cell increases and an oxidative process begins for lipids, proteins, and nucleic acids [47]. It has been accepted that iron and ROS may be increased, especially in tumor cells. The increased oxidative capacity of tumor cells may be effective in their growth. However, it is a contradictory opinion that increased ROS and iron content can increase ferroptosis. Toyokuni et al. reported the hypothesis that intracellular iron and thiol redox groups in tumor cells establish a balance for the cell to avoid ferroptosis. It is recognized that there are many more questions to be answered [25, 48]. Circulating non-heme iron can be transported bound and unbound to transferrin (Tf). Transferrin (Tf) is a glycoprotein with two high-affinity sites specific to ferric iron (Fe+3). When circulating Tf is fully saturated, iron can be transported independently of Tf ferric iron is reduced to ferro (Fe+2) iron by the presence of membranebound ferri reductases and taken into the cell by divalent metal transporter 1 (DMT1) [25, 49]. Most of the dietary heme iron is in the form of ferric iron. The absorption of inorganic iron from the lumen into the enterocyte in the duodenal villi is regulated in a very complex and molecularly controlled system. The first step in absorption is Fe+3 reduced to Fe+2 by ascorbate-dependent duodenal sitokrom *b* (DCYTB), a membranebound reductase. Ferrous iron is taken up into the enterocyte by DMT1 on the lumenfacing surface of the enterocyte. DMT1 is the most important molecule of nonheme iron intake. The synthesis of both DCYTB and DMT 1 is increased in iron deficiency [49–51]. Although intestinal absorption of heme iron (e.g., red meat) is effective for the human organism, it is by a mechanism that is not yet clearly understood. For heme absorption from the duodenum and upper jejunum, coordination of heme carrier protein 1 (HCP1) and heme responsive gene-1 is required [52, 53].

Under physiological conditions, the high-affinity Tf receptor 1 (TfR1) on the cell surface can bind two Fe(III). The Tf-Fe+2-TfR1 complex is transported into cells via endocytosis to form endosomes. Endosomes release iron from the complex under acidic conditions. Free ferric iron is reduced to ferrous iron and then transported into the cytoplasm by DMT1. Endosomes containing the Apo-Tf-TfR1 complex return to the surface of the cells and ferrous iron becomes part of the labile iron pool (LIP) in preparation for the next

*Ferroptosis: Can Iron Be the Downfall of a Cell? DOI: http://dx.doi.org/10.5772/intechopen.101426*

recycling [49, 50] Iron inside the cell can be stored in ferritin, transferred via ferroportin (FPN), or used in synthesis pathways [44]. Most of the intracellular iron is used for heme and iron-containing proteins, especially mitochondrial iron-sulfur-containing proteins (Fe-S) and iron-dependent enzymes. FPN is the only molecule known to transport iron out of the cell [54]. Iron transferred out of the cell via FPN is in ferrous form. In this transfer, ferrous iron is oxidized by extracellular ferroxidase and converted to ferric iron. The free ferric iron that becomes free is bound to Tf, forming Tf-Fe+2 complexes, and iron is transported to other cells. The iron that is not transported out of the cell and not used in the cell is stored by binding to ferritin. Ferritin is a heterodimer consisting of 24 subunits as ferritin heavy chain 1 (FTH1) and ferritin light chain (FTL). FTH is the domain that binds iron molecules, and FTL can play a role in electron transport. FTH can bind 4500 iron atoms in the ferric form [55]. Iron release from ferritin is also controlled under physiological conditions [56]. In recent studies, nuclear receptor coactivator 4 (NCOA4) mediated ferrophagy has been shown to induce iron release from ferritin. NCOA4 binds to ferritin and transports it to the lysosome, where ferric iron is decomposed and released [57]. The amount of iron in the cell increases. Therefore, it is postulated that NCOA4 mediated ferritinophagy can induce ferroptosis in the cell [58–61].

#### **6. Regulation of systemic iron**

Systemic iron level regulation is carried out through the FPN, which ensures the removal of iron from the cell. FRN is regulated both dependently and independently of hepcidin. In response to adequate systemic iron content, the liver secretes hepcidin into the systemic circulation. Hepcidin binds to FPN in the cell, causing a conformational change in the molecule. The modified FPN molecule is phosphorylated and ubiqutinized, then transported to the lysosome and inactivated by lysosomal enzymes. FPN can also be regulated independently of hepcidin. When there is not enough iron in the cell, FNP undergoes a similar conformational change and is again inactivated in the lysosome. In both cases, the removal of iron from the cell by the FPN is prevented [62].

### **7. Regulation of intracellular iron**

Cell internal iron homeostasis is regulated by iron regulatory protein 1, 2 (IRP1, IRP2) and iron-responsive elements (IREs) molecules. IRPs are proteins that can bind 5′,3′ (UTR) of the mRNAs of IREs. These proteins are those involved in iron uptake (e.g., DMT1, TfR1), iron sequestration (e.g., subunits of ferritin: FTH1, FTL), and iron export (e.g., FPN). When intracellular iron is deficient, IRPs bind to 5′ IREs of ferritin and FPN, and the translocation of these proteins is inhibited [63, 64]. Once iron demand is met, IRPs are degraded and these bonds are removed [63, 64]. Both cellular and systemic iron regulation is related to meeting iron needs. Systemic regulation is provided by the liver and hepcidin. The loss of binding activity of IRP1-IREs for cellular level iron regulation is related to the addition of 4Fe-4S. An E3 ligase complex linked to F-box and leucine-rich repeat protein 5 (FBXL5) drives ubiquitination and proteasomal degradation of IRP-2. FBXL5 requires sufficient iron and oxygen to remain stable. Other genes involved in iron metabolism are IREB2, FBXL5, TfR1, FTH1, and FTL [63–66].

It has been stated that the increase in iron level in the systemic circulation and intracellularly in vivo and in vivo conditions increases the susceptibility to ferroptosis. FPN decreases while Tf increases in ferroptosis-sensitive cells [58, 67]. Lysosomal high


*damage regulated autophagy modulator 3; FSP1, ferroptosis suppressor protein 1; GPX4, glutathione peroxydase 4; HSPB1, heat shock protein beta-1; Keap1, Keleh-like ECH-associated protein 1; MAPK, mitogen-activated protein kinase; MLKL, mixed lineage kinase domain-like protein; m-TOR, mammalian target of rapamycin; MVA, mevalonate; LC3, microtubule-associated protein 1 light chain 3; NCOA4, nuclear coactivator 4; NRF2, nuclear factor erythroid 2-related factor 2; PKC, protein kinase C; RIP, receptor-interacting serine/threonine kinase; ROS, reactive oxygen species; SAT1, spermidine/spermine N1-acetyltransferase 1; SLC7A11, solute carrier family 7 member 11; system, Xc-cysteine/glutamate transporter; TFEB, transcription factor EB; TFR1, transferrin receptor 1; TNF-R1, tumor necrosis factor R1.*

#### **Table 1.**

*Characteristics of ferroptosis.*

concentrations of iron can also prepare cells for ferroptosis [67]. Increased susceptibility to ferroptosis has been observed experimentally in mice with a high amount of iron in their diet and with increased extracellular matrix iron levels [68]. The heat shock protein family B member 1 (HSPB1) inhibits iron uptake via TfR1, reduces the level of iron into the cell, and inhibits ferroptosis by increasing the reduced form of GSH. HSPB1 also inhibits endocytosis and Trf1 reuptake by stabilizing the cortical actin cytoskeleton [69–71]. Both oxygenase 1 (HO-1) and phosphorylase kinase catalytic subunit gamma 2 (PHKG2) mediate ferroptosis when the intracellular iron level is increased [9, 72]. Almost all of the studies on this subject emphasize increased intracellular iron levels and ROS load to initiate ferroptosis. Until now, 3 basic processes that increase ROS in the cell due to iron have been reported: 1. by the Fenton reaction, which is inorganically not enzyme-catalyzed 2. via lipid autooxidation catalyzed by iron-containing enzymes 3. ROS formed by AA oxidation via iron-containing LOXs. However, how iron initiates and maintains ferroptosis and the process leading to cell death has not been fully elucidated (**Table 1**) [44].

#### **8. P62 and NRF2 in ferroptosis**

Nuclear factor erythroid 2-associated factor 2 (NRF2) is one of the proteins that create the most important antioxidant response in the cell against oxidative imbalance. Under normal conditions, it is preserved by Kelch-like ECH (erythroid cell-derived protein with CNC homology)-associated protein 1-mediated proteasomal degradation. NRF2 negatively regulates ferroptosis via the p62-keap1-NRF2 pathway. NRF2 and p62 competitively bind to Keap1 [73, 74]. Nrf2 plays a vital role in intracellular antioxidant balancing and activation of GPX4, in the re-synthesis of NADPH, 6PGD (phosphogluconate dehydrogenase, malic enzyme, and glucose 6-phosphate dehydrogenase)

#### *Ferroptosis: Can Iron Be the Downfall of a Cell? DOI: http://dx.doi.org/10.5772/intechopen.101426*

and glutathione synthesis, cysteine supply via system Xc (glutathione peroxidase 4, glutathione reductase) play a key role for many genes. Ferroptosis inducers facilitate the interaction between p62 and Keap1. This interaction inhibits Keap1. Inhibition of Keap1 prevents binding between Keap1 and NRF2. The interaction of Keap1 and NRF2 triggers the degradation of NRF2 [75, 76]. It leads to NRF2-mediated ferroptosis by downregulating genes involved in iron and ROS metabolisms. The most important of these are such as quinone oxidoreductase 1 (NQO1 and HO1) [75, 76].

NRF2 inhibits ferroptosis by increasing the expression of target genes involved in iron and ROS metabolism, such as NQO1 (NADPH Quinone Dehydrogenase 1) and HO1 (Heme oxygenase 1). NRF2-Keap1 pathway supports system Xc so NRF2 inhibits ferroptosis. It also negatively regulates ferroptosis by lowering intracellular reactive iron by gene regulation of Nrf2 ferritin (FTL/FTH) light chain and heavy chains, ferroportin (SLC40A1) subunit, and SLC7A11 component of Xc system. Nrf2 is also activated by oxidized lipids, which are also involved in the initiation of ligand-mediated transcription factor PPAR γ (peroxisome proliferator-activated receptor-gamma). Furthermore, a high NRF2 expression is associated with a worse overall survival rate in patients with glioma, and activation of the NRF2-Keap1 pathway supports system Xc so NRF2 inhibits ferroptosis [77]. The existence of studies reporting that NRF2 induces ferroptosis shows that there are still unanswered questions on this subject.

#### **9. Tumor suppressor protein P53 and ferroptosis**

P53 is a tumor suppressor that has been extensively studied. It has a tumorsuppressive effect by stopping metabolic cycles, mediating aging and apoptosis. It is involved in the cellular response to DNA damage, hypoxia, starvation, and oncogene activation. Activation of p53 ensures cell cycle slowdown at the low level of cellular stress, repair DNA damage, prevent ROS accumulation, and cell survival. However, severe cellular stress and damage induce a response of P53 to produce apoptosis and cell death [78]. On the one hand, p53 suppresses ferroptosis either through direct inhibition of DPP4 (dipeptidyl peptidase 4) activity [79] or through induction of CDKN1A/p21 (cyclin-dependent kinase inhibitor 1A) expression. On the other hand, 53 can increase ferroptosis by inhibiting the expression of SLC7A11 (solute transporter family 7 member 11) or by increasing the expression of SAT1 (spermidine/spermine N1-acetyltransferase 1) and GLS2 (glutaminase 2) [78–80].

It is accepted that the direction and intensity of the response of p53 are proportional to the level of stress to which the cell is exposed [80]. It is depleted by a mutation in many types of cancer and its anti-tumor effect is limited [81, 82]. Unlike nuclear p53, which acts as an autophagy-promoting transcription factor [82, 83], cytosolic p53 can block autophagy in response to nutrient starvation or mTOR inhibition [80, 83, 84]. These context-dependent roles of p53 in survival and death are regulated in a fine-tuned manner by its ubiquitination, phosphorylation, acetylation, and other modifications. Unlike nuclear p53, which functions as a transcription factor promoting autophagy, cytotic p53 (BCL-2 family (BAX [BCL2 associated X, apoptosis regulator] and BBC3/PUMA [BCL2 binding component 3]) can suppress autophagy in response to cellular starvation and mTOR inhibition [81–84]. p53 cellular response is regulated by ubiquitination, phosphorylation, acetylation, and other modifications [81].

Classic ferroptosis model and cell culture studies have revealed that P53 is associated with ferroptosis [79, 82, 84]. The researchers have found that 53 promotes ferroptosis due to transrepression of SLC7A11 expression in fibroblasts and some cancer cells

(human breast cancer MCF7) and human osteosarcoma (U2OS) [79, 85, 86]. P53 plays a role in ferroptosis cascades, which can cause cell survival or death. It can function prodeath or prosurvival at the transcriptional or post-translational level. Depending on the type or severity of stress that the cell is exposed to, it may contribute to apoptosis or autophagy [87, 88].

Inhibition of SLC7A11 expression, increased expression of SAT1 (spermidine/ Spermine N1-acetyltransferase 1), increased expression of GLS (Glutaminase) are required for p53-regulated ferroptosis [78]. SAT1 is a regulator of polyamine metabolism. Oxidative stress, inflammatory stimuli, and heat shock have been found to stimulate SAT1 activity. SAT1 is a transcriptional target of p53. An increase in SAT 1 does not change SLC7A11 and GPX4 activity but increases ALOX15 (arachidonate 15-lipoxygenase) activity [84]. Thus, the required antioxidant response remains insufficient despite increased lipid peroxidation.

Acetylation of K98 is crucial for p53-mediated ferroptosis. In particular, p53 3KR, an acetylation-defective mutant in which 3 lysine residues (at positions 117, 161, and 162) have been replaced by arginine residues, is highly effective in repressing SLC711A [85–87]. In contrast, p53 4KR98 (an acetylation-defective mutant in which an additional lysine is replaced at position 98) cannot reduce SLC711A expression [80].

Perhaps p53 3KR gains a ferroptosis-inducing capacity while p53 4KR loses it. In human cancers, wild-type p53 is degraded by high levels of the oncogenic E3 ubiquitinprotein ligase MDM2. Thus, inhibition of MDM2-dependent proteasomal degradation of p53 offers an attractive therapeutic strategy for cancer therapy [88]. Since it will not be inactivated in MDM2−/− cells, the p53 level increases. p53 has been shown to contribute to the cell death cascade, which can be termed ferroptosis, which can be reversible by ferrostatin 1 in MDM2−/− mouse embryos. However, another study showed that ferrostatin-1 alone could not prevent cell death caused by MDM2 deficiency [89–91].

The anti-ferroptosis activation of ferrostatin-1 and liproxstatin-1 (another widely-used ferroptosis inhibitor) are mediated through their reactivity as radical-trapping antioxidants rather than their potency as inhibitors of lipoxygenases [90, 91]. The acetylation levels of p53 are localized by six different histone acetyltransferase: 1. REBBP/CBP (CREB binding protein), 2. EP300/p300 (E1A binding protein P300), 3. KAT2B/PCAF (lysine acetyltransferase 2B), 4. KAT5/Tip60 (lysine acetyltransferase), 5. KAT8/MOF (lysine acetyltransferase 8), and 6. KAT6A/MOZ (lysine acetyltransferase 6A). The ability of these acetyltransferases to regulate ferroptosis remains unclear [88, 92, 93].

GSL2 (glutaminase 2) is a mitochondrial enzyme, the first step of glutamine catabolism, and an important regulator of ferroptosis [94]. It is known as a transcriptional target of p53. It is responsible for oxygen consumption and ATP production in cancer cells. It is also known to offer antioxidant support through the production of GSH [95]. While all this is expected for negative regulation of ferroptosis, it has been shown that glutaminase degradation inhibits ferroptosis in fibroblast cells [96]. More research is needed for the relationship between glutaminase, p53, and ferroptosis.

DPP4 (dipeptidyl peptidase-4) is the most important regulator of survival in the ferroptosis-related function of P53. Cells with p53 knockout or pharmacologically inhibited become more sensitive to type I inducer of ferroptosis (erastin and SAS). However, there is no difference in response to typeII ferroptosis inducer (RSL3 and FIN56).

However, DPP4 inhibitors (linagliptin, vildagliptin, and alogliptin) together with other protease inhibitors (doxycycline, ritonavir, atazanavir, VX-222, semagacestat) completely block erastin-induced cell death in p53-deficient cells [79].

Another mediator of p53, CDKN1A/p21 (cyclin-dependent kinase inhibitor 1A), inhibits apoptosis. In cystine deficiency in cancer cells, p53-mediated CDKN1A

expression delays ferroptosis. Again, inhibition of MDM2 by nutlin-3 increases expression of p53, which blocks the ferroptosis induced by loss of Xc function [82]. More comprehensive and detailed studies on p53 and ferroptosis are needed.

#### **10. Beclin-1 and ferroptosis**

Beclin-1 (Vps30/Atg6 in yeast) is a well-known regulator of autophagy primarily involved in the formation of the PtdIns3K complex, which is involved in activating autophagy. Beclin-1 is a critical regulator of ferroptosis that is independent of the formation of the PtdIns3K complex. The beclin-1 expression only affects ferroptosis induced by the system Xc-inhibitor. Knockdown of Beclin-1 by RNA interference (RNAi) blocks ferroptosis, whereas knockdown of Beclin-1 by gene transfection promotes ferroptosis in cancer cells in response to system Xc-inhibitors (for example, erastin, sulfasalazine, and sorafenib). In contrast, it does not affect erastin-, sorafenib-, or sulfasalazine-induced ferroptosis. Beclin-1 mandatory for ferroptosis induced by system Xc − inhibitor [97, 98].

It needs ATG5 (related to autophagy 5) and NCOA4 (nuclear receptor coactivator 4). ATG5 is part of an E3-like ligase that is critical for the lipidation of members of GABARAP (GABA type-A receptor-associated protein families) and MAP1LC3 (microtubule-associated protein 1 light chain 3) members. However, NCOA4 is a transporter receptor that mediates FT/ferritin degradation via selective ferritinophagy. Inhibits elastin-induced conversion of MAP1LC3B-I to MAP1LC3B-II by inhibition of Atg5. Furthermore, suppression of NCOA4 blocks the degradation of FT/ ferritin, resulting in suppression of ferroptosis. In contrast, knockdown of Beclin-1 does not affect the synthesis of lapidated MAP1LC3B and MAP1LC3B-positive points in ferroptosis. As a positive control in starvation-induced cells, knockdown of Beclin-1 stops the conversion of MAP1LC3B-I to MAP1LC3B-II. Significantly, the formation of a BECN1-PtdIns3K complex was observed in cancer cells only in response to starvation, but not to ferroptotic stimulus. These findings point to the regulatory roles of Beclin-1 in ferroptosis compared to induced autophagy [96–98].

#### **11. AMPK ferroptosis**

AMP-activated protein kinase (AMPK), a critical indicator of the cell's energy deficit, is activated through AMP binding, kinase phosphorylation, and other mechanisms. AMP-activated protein kinase (AMPK), a critical indicator of the cell's energy deficit, is activated through AMP binding, kinase phosphorylation, and other mechanisms. AMPK maintains the viability of the cell under energy stress. If this energy balance cannot be achieved, it leads the cell to apoptosis. AMPK exhibits various regulatory roles in lipid metabolism by mediating the phosphorylation of acetyl-CoA carboxylase as well as polyunsaturated fatty acid biosynthesis. AMPK has also been implicated in ferroptosis. The inhibitory effect of AMPK activation on ferroptosis does not include modulation of cystine uptake, iron metabolism autophagy, or mTORC1 signaling. Energy stress-mediated AMPK activation inhibits ferroptosis via mitochondria. The Loss of function of liver kinase B1 (LKB1) sensitizes mouse embryonic fibroblasts (MEFs) and human non-small cell lung carcinoma cell lines to ferroptosis. This LKB1-AMPK-ACC1 (ACC1—Acetyl-CoA carboxylase 1)-FAS (cell surface death receptor) axis has a vital role in regulating ferroptotic cell death [99].

A recent study also reported a supportive role of AMPK in the regulation of Beclin-1-mediated ferroptosis. Specifically, AMPK mediates the phosphorylation of Beclin-1 at Ser90/93/96. This is a prerequisite for the formation of the Beclin1- SLC7A11 complex in ferroptosis and subsequent lipid peroxidation. Inhibition of AMPK by siRNA or compound C reduces erastin-induced Beclin-1 phosphorylation at S93/96, thus inhibiting the formation of Beclin-1-SLC7A11 complex formation and subsequent ferroptosis. Thus, it is clear that Beclin-1 contributes to the core molecular machinery and signaling pathways involved in ferroptosis [100, 101]. The mechanisms of AMPK-mediated regulatory ferroptosis need further investigation.

#### **12. Ataxia-telangiectasia-mutated kinase in ferroptosis**

Ataxia-telangiectasia mutated kinase (ATM) is a crucial kinase for DNA damage responses. P53 is one of its sub-targets, which plays a decisive role in the regulation of ferroptosis, which activates its role in ferroptosis [102]. Genetic or pharmacological inhibition of ATM reduces intracellular labile iron by increasing FPN, FTH1, and FTL. Ataxia-telangiectasia mutated kinase (ATM) is a crucial kinase for DNA damage responses. P53 is one of its sub-targets, which plays a decisive role in the regulation of ferroptosis, which activates its role in ferroptosis [103]. Genetic or pharmacological inhibition of ATM reduces intracellular labile iron by increasing FPNand FTH and FTL. It relies on the transcriptional activity and nuclear translocation of metal regulatory transcription factor 1 (MTF1) upon TM inhibition. Under conditions of ATM inhibition, nuclear translocation of MTF1 is increased, resulting in changes in ferritin (FTH1) and ferroportin (FPN) expression, and the amount of intracellular unstable iron is reduced to prevent ferroptosis [104].

#### **13. Iron and ferroptosis**

The iron homeostasis in both manners is regulated by iron. In systemic iron regulation, the level of iron is sensed by the liver and the liver secretes the hormone hepcidin according to iron abundance. At the cellular iron level, the loss of IRP1-IREs binding activity depends on the insertion of 4Fe–4S cluster. As for the IRP2, a newly discovered FBXL5-dependent E3 ligase complex catalyzes the ubiquitination and proteasomal degradation of IRP2, while keeping the stability of FBXL5 requires iron and oxygen IREB2, the main regulator of iron metabolism upon inhibition, reduces sensitivity to ferroptosis. Since iron metabolism is also affected by autophagy, it also regulates ferroptosis in many ways [58]. Ferritinophagy is the autophagy of selective ferritin, in which ferritin is recognized by the specific transport receptor NCOA4, which directs it to autophagosomes for lysosomal degradation. This lysosomal degradation of ferritin releases iron and thus increases ferroptosis susceptibility [45].

Apart from ferritin, HSPB1 and CISD1 are other proteins that affect ferroptosis susceptibility. In addition, heme oxygenase 1 (HO-1) and phosphorylase kinase catalytic subunit gamma 2 (PHKG2) mediate ferroptosis by regulating the abundance of iron [9, 72]. ROS accumulation initiated by labile iron in the cell occurs in three known ways, respectively. 1. non-enzymatic Fenton reaction; 2. ROS accumulated by lipid autooxidation catalyzed by iron-containing enzymes; 3. ROS accumulated by oxidation of arachidonic acid (AA) by lipid peroxidases (LOX). Although it is known that iron is an essential element in ferroptosis, it is not fully understood how iron regulates ferroptosis.

### **14. Biomarkers of ferroptosis**

Prostaglandin-endoperoxide synthase 2 (PTGS2) increases with the formation of lipid peroxides and decreases in nicotinamide adenine dinucleotide phosphate (NADPH) [9, 32]. Moreover, the increase in PTGS2 cannot be suppressed by PTGS inhibitors. Malondialdehyde (MDA), the end product of lipid peroxidation, also increases. GPX4 protects cells against ferroptosis by catalyzing GSH and toxic PE-AA-OOH to oxidize GSH (GSSG) and non-toxic PE-AA-OH. GSSG is then converted to GSH by GSH reductase (GR) in the presence of NADPH. Therefore, NADPH, a coenzyme of GR, plays a vital role in maintaining the abundance of intracellular GSH. Furthermore, the basal NADPH abundance of a given cell is negatively correlated with ferroptosis susceptibility. NADPH necroptosis can establish a link between ferroptosis and GSH (**Figure 1**) [9, 32].

**Figure 1.**

*ROS (reactive oxygen space) and ferroptosis.*

## **15. Ferroptosis and other forms of cell death**

Unlike ferroptosis, apoptosis, necrosis, and autophagy decreased mitochondrial volume, increased mitochondrial membrane density, reduction in mitochondrial cristae, and rupture of the outer membrane are observed. Also, ferroptosis cannot be stopped by inhibitors of apoptosis, necrosis, and autophagy [8, 9, 27]. Compared to ferroptosis, it may show common features with other regulated cell deaths. Although ferroptosis does show mitochondrial differences, it cannot be entirely attributed to it. The amount of mitochondrial ROS does not change in ferroptosis exposed to erastin. Moreover, ferroptosis also occurs in cells lacking a mitochondrial electron transport chain [8, 14, 105].

### **16. Ferroptosis and oxytosis**

Oxidative glutamate toxicity leads to glutathione depletion by inhibiting cystine uptake via exogenous glutamate, the Xc system (cystine/glutamate antiporter).

Activation of lipoxygenase opens up cGMP-gated channels that allow reactive oxygen species production and extracellular calcium influx. In oxytosis paradigms in neuronal cells, mitochondrial disorders are mediated through mitochondrial transactivation of pro-apoptotic protein (BID). Upon translocation to mitochondria, BID mediates loss of mitochondrial integrity and function and deleterious translocation of mitochondrial AIF to the nucleus. Induced by stress and independently of mitochondrial death. In neuronal cells, ROS-induced transactivation of BID into mitochondria links both oxytosis and ferroptosis pathways and leads to irreversible morphological and functional damage. MMP (Matrix metallo proteinases) loss, reduction of ATP levels, and mitochondrial ROS generation are associated with the apoptosis-inducing factor (AIF). The BID inhibitor BI-6c9 and the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 can block these deadly pathways upstream of mitochondrial disorders. The analogy of ferroptosis and oxytosis seems to be the most promising for treatment options, especially in diseases related to iron accumulation such as neurodegenerative diseases, stroke, and reperfusion injury [106–108].

### **17. Ferroptosis and necroptosis**

They used ACSL4 as a ferroptosis susceptibility marker and a Mixed Lineage Kinase domain-like (MLKL) marker for necroptosis. Interestingly, ACSL4 deficiency led to an increase in MLKL, and loss of MLKL increased the cells' sensitivity to ferroptosis. When one cell death pathway is inhibited, it evolves into the other pathway. It has been demonstrated that ferroptosis and necroptosis are different forms of cell death. They used ACSL4 as a ferroptosis susceptibility marker and a mixed lineage kinase domainlike (MLKL) marker for necroptosis. Interestingly, ACSL4 deficiency led to an increase in MLKL, and loss of MLKL increased the cells' sensitivity to ferroptosis. When one cell death pathway is inhibited, it evolves into the other pathway [15, 16, 109, 110].

#### **18. Ferroptosis and autophagy**

Ferritinophagy is the autophagic process of ferritin mediated by NCOA4. NCOA4 binds to FTH1 in autophagosomes during low intracellular iron and then autophagosomes are sent to lysosomes for ferritin degradation. Similarly, autophagy is the death of the cell by breaking down organelles and proteins in the cell with phagosomes and lysosomes [57]. Cell death can also be blocked by the inhibition of ferritinophagy in aged cells [60]. Ferritinophagy and unstable iron increase in fibroblast culture and cancer cells accelerated cell death [59]. Increased ferritinophagy in liver fibrosis and erastininduced ferroptosis follow the same process [61]. Ferritinophagia has been reported at the onset of ferroptosis [100]. However, it differs from ferritinophagy in that specific autophagy inhibitors fail to rescue ferroptosis [11].

#### **19. Conclusion**

Ferroptosis differs from other forms of regulated cell death. It requires an unstable form of intracellular iron. It is associated with the increased reactive oxidative load. The susceptibility to ferroptosis differs from cell to cell. It presents new research areas for the treatment of cancer, circulatory diseases, and degenerative neurological disorders.

*Ferroptosis: Can Iron Be the Downfall of a Cell? DOI: http://dx.doi.org/10.5772/intechopen.101426*

### **Abbreviations**



## **Author details**

Asuman Akkaya Fırat Biochemistry Department, Fatih Sultan Mehmet (F.S.M) Training and Research Hospital, Health Sciences University (S.B.U), İstanbul, Turkey

\*Address all correspondence to: asumanfirat44@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Ferroptosis: Can Iron Be the Downfall of a Cell? DOI: http://dx.doi.org/10.5772/intechopen.101426*

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Section 3
