*2.3. Iron and Ferroptosis*

In the human body, iron metabolism is regulated by means of a perfectly adjusted balance between plasma proteins. They are associated with the transport, absorption and recycling of iron, in order to avoid the accumulation of iron, which is highly harmful and reactive in tissues. Figure 3 shows several aspects of human iron metabolism. Biochemically, iron is capable of accepting and donating electrons, interconverting between the ferric (Fe3<sup>+</sup>) and ferrous (Fe2<sup>+</sup>) forms, which are both found in the human body. The Fe3+/Fe2<sup>+</sup> redox potential participates in a large number of protein complexes, especially those that involve oxygen reduction for adenosine triphosphate (ATP) synthesis and the reduction of DNA precursors. Iron is also a necessary component in the formation of molecules that bind and transport oxygen (hemoglobin and myoglobin) and for the activities of cytochrome enzymes, as well as in many enzymes that perform the redox process, functioning as electron carriers [83–85].

Physiologically, iron that will be distributed to tissues needs to bind to transferrin (apotransferrin), giving rise to holotransferrin [87]. The distribution of iron to the tissues occurs through the endocytosis of holotransferrin, mediated by binding to transferrin receptor type 1 (TFR1) and type 2 (TFR2) [88]. After endocytosis, the holotransferrin–TFR1 complex is mobilized to the endosomes. In the acid environment of the endosome, Fe3<sup>+</sup> is released from TF and converted to Fe2<sup>+</sup> by oxidation-reduction, by six-transmembrane epithelial antigen of the prostate 3 (STEAP3) and then exported into cytosol by divalent metal transporter 1 (DMT-1). Iron can be stored in ferritin/hemosiderin or remain labile [89].

The labile iron pool (LIP), composed mostly of Fe2<sup>+</sup>, is a pool of chelable iron and active redox present in the cell; it can be present in mitochondria, lysosomes, cytosol and in the nucleus [90]. The concentration of LIP is essentially regulated by the absorption, use, distribution and export of iron in the cell and in the body. Labile iron has high chemical reactivity and exhibits high cytotoxic potential. Cytotoxicity is associated with the fact that labile iron catalyzes the formation of hydroxyl radicals (OH·) derived from hydrogen peroxide (H2O2) through the Fenton reaction (Figure 1). Moreover, H2O2 has a lower capacity to react with molecules, while OH· generated from the iron-dependent Fenton reaction has high reactivity with biological molecules, such as proteins and DNA, and generates lipid (hydro) peroxidation in ferroptosis [12–17].

Indeed, glutathione has a high affinity with Fe2<sup>+</sup> and the major component of LIP in cytosol is presented as the glutathione-Fe2<sup>+</sup> conjugates [90]. This is important because the decrease in intracellular glutathione increases the concentration of Fe2<sup>+</sup>, facilitating the Fenton reaction [90–93]. The storage of labile iron in ferritin serves to prevent its high reactivity, avoiding the generation of reactive species [94]. The ferritin structure is formed by 24 subunit-composed chains both light (L) and heavy (H) with a spherical "shell" shape, which accommodates about 4500 iron atoms. H-ferritin contains a ferroxidase, which oxidizes Fe2<sup>+</sup> to Fe3<sup>+</sup>, to store iron inside the nucleus. When necessary, iron stocks are mobilized and exported by ferroportin (FPN). This process is downregulated by hepcidin [83–85].

**Figure 3.** Human Iron metabolism. Iron concentration in the body is maintained through diet and the recycling of senescent erythrocytes. The daily diet provides approximately 1–2 mg of iron. Enterocytes, present in the duodenum and in the proximal portion of the jejunum, can absorb both ferrous iron (heme iron) and ferric iron (non-heme). However, it is necessary to reduce ferric iron to ferrous iron, by apical ferric reductase enzymes, such as enzyme duodenal cytochrome b (Dcytb), for absorption to occur. Then, iron is transported by the divalent metal type transporter-1 (DMT-1) and stored inside the cell [86]. Ferrous iron from the diet is internalized by the heme-1 carrier protein (HCP) in cells, where it is stored as hemosiderin and/or ferritin, to prevent the Fenton reaction. Physiologically, iron stores are mobilized from intracellular to the extracellular by ferroportin (FPN) when the serum iron is low. Iron released in its ferrous state is oxidized to ferric iron and binds to serum apotransferrin to be transported through the body, giving rise to holotransferrin. This oxidation reaction occurs through the action of oxidase enzymes: hephestine present in enterocytes, ceruloplasmin present in hepatocytes macrophages. The distribution of iron to the tissues occurs through the endocytosis of holotransferrin, mediated by the binding to transferrin receptors type 1 (TFR1) and type 2 (TFR2). Ferroportin mediates the efflux of iron within cells, maintaining systemic iron homeostasis. This process is negatively regulated by hepcidin, which promotes ferroportin endocytosis and then proteolysis in lysosomes by induced ubiquitination [83]. The recycling of iron by macrophages occurs through the phagocytosis of senescent erythrocytes and hemoglobin and the heme group of intravascular hemolysis. Once internalized in the macrophage, the heme group releases ferrous iron through the activity of the enzyme heme oxygenase, which can be exported to the extracellular medium by ferroportin or stored as ferritin [83,84].

The intracellular iron content is regulated by the iron regulatory proteins (IRP1 and IRP2) and the iron-responsive element (IRE) [95,96]. IRPs can bind to RNA stem-loops containing an IRE in the untranslated region (UTR), affecting the translation of target Mrna: 3- UTR of H-ferritin mRNA and in the 5-UTR of TFR1 mRNA. In response to cellular iron demand IRE/IRP interaction promotes TFR1 mRNA stability and inhibits H-ferritin translation, thus modulating cellular iron uptake and storage. Overexpression of both TF and TFR1 sensitizes cells to ferroptosis by enhancing iron uptake; on the other hand, silencing TFR1 can inhibit erastin-induced ferroptosis [16,97–99]. In addition, anti-TfR1 antibodies identify tumor ferroptotic cells from different tissues [100].

Autophagy in fibroblasts leads to erastin-induced ferroptosis through the degradation of ferritin and induction of TfR1 expression [101]. On the other hand, cellular senescence has been associated with intracellular iron accumulation and impaired ferritinophagy [102]. Ferritinophagy induces ferroptosis by increasing the nuclear receptor coactivator 4 (NCOA4), followed by an increase in ferritin degradation in the phagolysosome and release of Fe2<sup>+</sup> (labile iron pool) in the cytoplasm. In fact, some authors consider ferroptosis to be a type of autophagy [103–107].

Another molecule involved in ferroptosis is the sigma-1 receptor (S1R), which protects hepatocellular carcinoma cells against ferroptosis. S1R regulates ROS accumulation via Nrf2. Knockdown of S1R blocks the expression of GPx4 and HO-1. Moreover, knockdown of S1R significantly increases Fe2<sup>+</sup> levels and MDA production in HCC cells treated with erastin and sorafenib, as well as the upregulation of H-ferritin chain and TRF1 [108]. In addition, it has been shown that heat shock protein β-1 (HSPβ1) is a negative regulator of ferroptotic cancer cell death, and erastin stimulates heat shock factor 1 (HSF1)-dependent HSPβ1. Knockdown of HSF1 and HSPβ1 enhances erastin-induced ferroptosis, whereas heat shock pretreatment and overexpression of HSPβ1 inhibits erastin-induced ferroptosis by protein kinase C. Moreover, the increase in cellular iron in HSPβ1 knockdown cells has been associated with increased expression of TFR1 and a mild decrease in the expression of the H-ferritin chain [109]. HSPA5, an endoplasmic reticulum (ER)-sessile chaperone, was shown to bind and stabilize GPx4, thus indirectly counteracting lipid peroxidation in ferroptosis [97].
