*2.1. Lipid Peroxidation and Ferroptosis*

Lipid peroxidation is the trigger for the activation of ferroptosis [13,18]. Lipid peroxides (PL-OOH), mainly lipid hydroperoxides (L-OOH), have the ability to cause damage to the lipid bilayer of the plasma membrane due to the accelerated oxidation of the membrane lipids which leads to ferroptosis. The increase in the concentration of lipid peroxides can alter the structure and function of nucleic acids and proteins, as well as the Michael acceptors and aldehydes. In fact, it can generate additional toxicity due to its degradation products [19–22]. The cellular lipids include thousands of lipid species that vary in quantity, intra- and extracellular distribution, functions and cell type [8]. Thus, the higher the concentration of free polyunsaturated fatty acids (PUFAs) in the cell, the greater the damage caused by lipid hydroperoxidation and the extent of ferroptosis, which can vary among diseases and organs/tissues [4,8,17].

PUFAs are good substrates for autoxidation because the C–H bonds of the methylene groups flanked by C-C double bonds are among the weakest C–H bonds known [19–22]. The structure of the PUFA molecule contains bis-allyl hydrogen atoms that can be abstracted. Then, there is a rearrangement of the resonance radical structure, with subsequent addition of molecular oxygen, giving rise to the peroxyl radical and the formation of the primary molecular product, lipid hydroperoxide (L-OOH). Soon after, the cleavage of the L-OOH molecule occurs, giving rise to highly electrophilic secondary oxidation products, including epoxy, oxo- or aldehyde groups, which are highly reactive and toxic to membranes and cells [8,23].

First, PUFAs are esterified with membrane phospholipids, such as phosphatidyl ethanolamine (PE). The esterification reaction is catalyzed by acyl-CoA synthetase long-chain family member 4 (ACSL4), which binds coenzyme A to long-chain PUFAs, which can then be used for esterification of lysophospholipids by lysophosphatidylcholine acyltransferase 3 (LPCAT3); the substrates can undergo peroxidation resulting in the formation of arachidonoyl (AA) and adrenoyl (AdA) acids, which can lead to ferroptosis. Suppression of the ACSL4 enzyme inhibits ferroptosis by depleting the substrates for lipid peroxidation [24,25].

The PUFA oxidation process that leads to ferroptosis can occur enzymatically or non-enzymatically [26]. The non-enzymatic oxidation process occurs through ROS and hydroxyl radical, from the Fenton reaction. This process is both non-selective and non-specific. Thus, oxidation rates are proportional to the number of readily abstractable bis-allyl hydrogens in the PUFA molecule, resulting in the accumulation of a highly diversified pattern of oxidation products with the predominance of oxygenated PUFA-PLs with 6, 5, 4, 3 and 2 double bonds [8,19]. Enzymatic oxidation of PUFAs occurs through lipoxygenases (LOXs) [27]. LOXs are dioxigenases containing iron in their catalytic region that promote the dioxigenation of polyunsaturated fatty acids containing at least two isolated cis-double bonds. In humans, there are different isoforms of LOX (5-LOX, 12S-LOX, 12R-LOX, 15-LOX-1, 15-LOX-2 and eLOX3) [19,27].

Membrane ester lipids are cleaved by cytosolic phospholipase A2 in different fatty acids: arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Oxygenation by cyclooxygenases (COXs) generates prostanglandins-G (PGG2, PGG3 and PGG4, respectively). However, oxygenation by LOX generates doubly and triply oxygenated (15-hydroperoxy)-diacylated PE species [28–31]. Oxidation induced by 15-LOX is selective and specific, occurring preferably in arachidonic acid-phosphatidylethanolamine (AA-PE) or adrenoyl acid (AdA)-PE. The product of this oxidation is 15-hydroperoxy-arachidonic acid-phosphatidylethanolamines (15-HOO-AA-PEs) or 15-hydroperoxy-adrenoyl acid-phosphatidylethanolamines (15-HOO-AdA-PEs) (Figure 1) [28–31]. The catalytic activity 15-LOX is dependent on the pro-ferroptotic PEBP1 protein [32].

Stoyanovsky et al. [33] showed that the ferroptosis process includes two stages: (i) selective and specific enzymatic production of 15-HOO-AA-PE by 15-LOX; (ii) oxidative cleavage of these initial HOO derivatives to proximate electrophiles capable of interacting with protein targets to cause the formation of pores in plasma membranes, or to rupture them. The two types of oxidatively truncated products can be formed from 15-HOO-AA-PE with the carbonyl function either on the shortened AA-residue esterified into PE, or on the leaving aldehyde.

In addition, tocopherols and tocotrienols suppress LOX and protect against ferroptosis [24]. On the other hand, ferrostatins inhibit ferroptosis by efficiently scavenging free radicals in lipid bilayers [34].

Recently, Zou et al. [35] have shown that cytochrome P450 oxidoreductase (POR) is a key mediator in the induction of ferroptosis in cells that exhibit intrinsic and induced susceptibility to ferroptosis by enabling membrane polyunsaturated phospholipid peroxidation. POR depletion suppressed arachidonic acid-induced sensitivity to ML210/RSL3 in a dose-dependent manner. In addition to suppressing PUFA-induced ferroptosis susceptibility, POR depletion by constitutive or inducible knockout also compromised the intrinsic ferroptosis sensitivity in ccRCC cells 786-O and 769-P.
