Dioxetane Synthesis Essay

Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Seville, Prof García Gonzales s/n., 41012 Seville, Spain

Copyright © 2014 Antonio Ayala et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Lipid peroxidation can be described generally as a process under which oxidants such as free radicals attack lipids containing carbon-carbon double bond(s), especially polyunsaturated fatty acids (PUFAs). Over the last four decades, an extensive body of literature regarding lipid peroxidation has shown its important role in cell biology and human health. Since the early 1970s, the total published research articles on the topic of lipid peroxidation was 98 (1970–1974) and has been increasing at almost 135-fold, by up to 13165 in last 4 years (2010–2013). New discoveries about the involvement in cellular physiology and pathology, as well as the control of lipid peroxidation, continue to emerge every day. Given the enormity of this field, this review focuses on biochemical concepts of lipid peroxidation, production, metabolism, and signaling mechanisms of two main omega-6 fatty acids lipid peroxidation products: malondialdehyde (MDA) and, in particular, 4-hydroxy-2-nonenal (4-HNE), summarizing not only its physiological and protective function as signaling molecule stimulating gene expression and cell survival, but also its cytotoxic role inhibiting gene expression and promoting cell death. Finally, overviews of in vivo mammalian model systems used to study the lipid peroxidation process, and common pathological processes linked to MDA and 4-HNE are shown.

This review paper is dedicated to
Dr. Alberto Machado

1. Lipids Overview of Biological Functions

Lipids Are Classically Divided into Two Groups: Apolar and Polar. Triglycerides (apolar), stored in various cells, but especially in adipose (fat) tissue, are usually the main form of energy storage in mammals [1, 2]. Polar lipids are structural components of cell membranes, where they participate in the formation of the permeability barrier of cells and subcellular organelles in the form of a lipid bilayer. The major lipid type defining this bilayer in almost all membranes is glycerol-based phospholipid [3]. The importance of the membrane lipid physical (phase) state is evidenced by the fact that lipids may control the physiological state of a membrane organelle by modifying its biophysical aspects, such as the polarity and permeability. Lipids also have a key role in biology as signaling molecules.

Lipids as Signaling Molecules. The main enzymes that generate lipid signaling mediators are lipoxygenase, which mediate hydroperoxyeicosatetraenoic acids (HPETEs), lipoxins, leukotrienes, or hepoxilins biosynthesis after oxidation of arachidonic acid (AA) [4, 5], cyclooxygenase that produces prostaglandins [4], and cytochrome P-450 (CYP) which generates epoxyeicosatrienoic acids, leukotoxins, thromboxane, or prostacyclin [4]. Lipid signaling may occur via activation of a variety of receptors, including G protein-coupled and nuclear receptors. Members of several different lipid categories have been identified as potent intracellular signal transduction molecules. Examples of signaling lipids include (i) two derived from the phosphatidylinositol phosphates, diacylglycerol (DAG) and inositol phosphates (IPs). DAG is a physiological activator of protein kinase C [6, 7] and transcription factor nuclear factor-kB (NF-κB), which promotes cell survival and proliferation. Diacylglycerol also interacts indirectly with other signalling molecules such as small G proteins [8]. IPs are a highly charged family of lipid-derived metabolites, involved in signal transduction that results in activation of Akt, mTOR [9], and calcium-homeostasis [10, 11]; (ii) sphingosine-1-phosphate, a sphingolipid derived from ceramide that is a potent messenger molecule involved in regulating calcium mobilization, migration, adhesion, and proliferation [12–14]; (iii) the prostaglandins, which are one type of fatty-acid derived eicosanoid involved in inflammation [15, 16] and immunity [17]; (iv) phosphatidylserine, a phospholipid that plays an important role in a number of signaling pathways, includes kinases, small GTPases, and fusogenic proteins [18]; (v) the steroid hormones such as estrogen, testosterone, and cortisol, which modulate a host of functions such as reproduction, metabolism, stress response, inflammation, blood pressure, and salt and water balance [19].

2. Lipids Damage by Reactive Oxygen Species

One of the consequences of uncontrolled oxidative stress (imbalance between the prooxidant and antioxidant levels in favor of prooxidants) is cells, tissues, and organs injury caused by oxidative damage. It has long been recognized that high levels of free radicals or reactive oxygen species (ROS) can inflict direct damage to lipids. The primary sources of endogenous ROS production are the mitochondria, plasma membrane, endoplasmic reticulum, and peroxisomes [20] through a variety of mechanisms including enzymatic reactions and/or autooxidation of several compounds, such as catecholamines and hydroquinone. Different exogenous stimuli, such as the ionizing radiation, ultraviolet rays, tobacco smoke, pathogen infections, environmental toxins, and exposure to herbicide/insecticides, are sources of in vivo ROS production.

The two most prevalent ROS that can affect profoundly the lipids are mainly hydroxyl radical () and hydroperoxyl (). The hydroxyl radical (HO) is a small, highly mobile, water-soluble, and chemically most reactive species of activated oxygen. This short-lived molecule can be produced from O2 in cell metabolism and under a variety of stress conditions. A cell produces around 50 hydroxyl radicals every second. In a full day, each cell would generate 4 million hydroxyl radicals, which can be neutralized or attack biomolecules [21]. Hydroxyl radicals cause oxidative damage to cells because they unspecifically attack biomolecules [22] located less than a few nanometres from its site of generation and are involved in cellular disorders such as neurodegeneration [23, 24], cardiovascular disease [25], and cancer [26, 27]. It is generally assumed that in biological systems is formed through redox cycling by Fenton reaction, where free iron (Fe2+) reacts with hydrogen peroxide (H2O2) and the Haber-Weiss reaction that results in the production of Fe2+ when superoxide reacts with ferric iron (Fe3+). In addition to the iron redox cycling described above, also a number of other transition-metal including Cu, Ni, Co, and V can be responsible for formation in living cells (Figure 1).

Figure 1: Fenton and Haber-Weiss reaction. Reduced form of transition-metals (Mn) reacts trough the Fenton reaction with hydrogen peroxide (H2O2), leading to the generation of . Superoxide radical () can also react with oxidized form of transition metals (M(n+1)) in the Haber-Weiss reaction leading to the production of Mn, which then again affects redox cycling.

The hydroperoxyl radical () plays an important role in the chemistry of lipid peroxidation. This protonated form of superoxide yields H2O2 which can react with redox active metals including iron or copper to further generate through Fenton or Haber-Weiss reactions. The is a much stronger oxidant than superoxide anion-radical and could initiate the chain oxidation of polyunsaturated phospholipids, thus leading to impairment of membrane function [28–30].

2.1. Lipid Peroxidation Process

Lipid peroxidation can be described generally as a process under which oxidants such as free radicals or nonradical species attack lipids containing carbon-carbon double bond(s), especially polyunsaturated fatty acids (PUFAs) that involve hydrogen abstraction from a carbon, with oxygen insertion resulting in lipid peroxyl radicals and hydroperoxides as described previously [31]. Glycolipids, phospholipids (PLs), and cholesterol (Ch) are also well-known targets of damaging and potentially lethal peroxidative modification. Lipids also can be oxidized by enzymes like lipoxygenases, cyclooxygenases, and cytochrome P450 (see above, lipid as signaling molecules). In response to membrane lipid peroxidation, and according to specific cellular metabolic circumstances and repair capacities, the cells may promote cell survival or induce cell death. Under physiological or low lipid peroxidation rates (subtoxic conditions), the cells stimulate their maintenance and survival through constitutive antioxidants defense systems or signaling pathways activation that upregulate antioxidants proteins resulting in an adaptive stress response. By contrast, under medium or high lipid peroxidation rates (toxic conditions) the extent of oxidative damage overwhelms repair capacity, and the cells induce apoptosis or necrosis programmed cell death; both processes eventually lead to molecular cell damage which may facilitate development of various pathological states and accelerated aging. The impact of lipids oxidation in cell membrane and how these oxidative damages are involved in both physiological processes and major pathological conditions have been analysed in several reviews [32–35].

The overall process of lipid peroxidation consists of three steps: initiation, propagation, and termination [31, 36, 37]. In the lipid peroxidation initiation step, prooxidants like hydroxyl radical abstract the allylic hydrogen forming the carbon-centered lipid radical (). In the propagation phase, lipid radical () rapidly reacts with oxygen to form a lipid peroxy radical () which abstracts a hydrogen from another lipid molecule generating a new (that continues the chain reaction) and lipid hydroperoxide (LOOH). In the termination reaction, antioxidants like vitamin E donate a hydrogen atom to the species and form a corresponding vitamin E radical that reacts with another forming nonradical products (Figure 2). Once lipid peroxidation is initiated, a propagation of chain reactions will take place until termination products are produced. Review with extensive information regarding the chemistry associated with each of these steps is available [31].

Figure 2: Lipid peroxidation process. In Initiation, prooxidants abstract the allylic hydrogen forming the carbon-centered lipid radical; the carbon radical tends to be stabilized by a molecular rearrangement to form a conjugated diene (step 1). In the propagation phase, lipid radical rapidly reacts with oxygen to form a lipid peroxy radical (step 2) which abstracts a hydrogen from another lipid molecule generating a new lipid radical and lipid hydroperoxide (step 3). In the termination reaction, antioxidants donate a hydrogen atom to the lipid peroxy radical species resulting in the formation of nonradical products (step 4).

2.2. Lipid Peroxidation Products

Lipid peroxidation or reaction of oxygen with unsaturated lipids produces a wide variety of oxidation products. The main primary products of lipid peroxidation are lipid hydroperoxides (LOOH). Among the many different aldehydes which can be formed as secondary products during lipid peroxidation, malondialdehyde (MDA), propanal, hexanal, and 4-hydroxynonenal (4-HNE) have been extensively studied by Esterbauer and his colleagues in the 80s [38–49]. MDA appears to be the most mutagenic product of lipid peroxidation, whereas 4-HNE is the most toxic [50].

MDA has been widely used for many years as a convenient biomarker for lipid peroxidation of omega-3 and omega-6 fatty acids because of its facile reaction with thiobarbituric acid (TBA) [48, 51]. The TBA test is predicated upon the reactivity of TBA toward MDA to yield an intensely colored chromogen fluorescent red adduct; this test was first used by food chemists to evaluate autoxidative degradation of fats and oils [52]. However, the thiobarbituric acid reacting substances test (TBARS) is notoriously nonspecific which has led to substantial controversy over its use for quantification of MDA from in vivo samples. Several technologies for the determination of free and total MDA, such gas chromatography-mass spectrometry (GC-MS/MS), liquid chromatography-mass spectrometry (LC-MS/MS), and several derivatization-based strategies, have been developed during the last decade [53]. Because MDA is one of the most popular and reliable markers that determine oxidative stress in clinical situations [53], and due to MDA’s high reactivity and toxicity underlying the fact that this molecule is very relevant to biomedical research community.

4-HNE was first discovered in 60s [54]. Later, in 80s 4-HNE was reported as a cytotoxic product originating from the peroxidation of liver microsomal lipids [40]. 4-Hydroxyalkenals produced in the course of biomembrane lipids peroxidation, elicited either by free radicals or by chemicals, might exert a genotoxic effect in humans [55]. The 4-hydroxyalkenals are the most significant products because they are produced in relatively large amounts, and they are very reactive aldehydes that act as “second messengers of free radicals.” In particular 4-HNE, which has been subjected to intense scientific scrutiny in 90s [49], is considered as “one of the major toxic products generated from lipid peroxides” [49]. 4-HNE high toxicity can be explained by its rapid reactions with thiols and amino groups [56]. Reactive aldehydes, especially 4-HNE, act both as signaling molecules (see below 4-HNE as signaling molecule) and as cytotoxic products of lipid peroxidation causing long-lasting biological consequences, in particular by covalent modification of macromolecules (see below 4-HNE biomolecular adducts). 4-HNE is considered as “second toxic messengers of free radicals,” and also as “one of the most physiologically active lipid peroxides,” “one of major generators of oxidative stress,” “a chemotactic aldehydic end-product of lipid peroxidation,” and a “major lipid peroxidation product” [57]. Thus, it is not a surprise that 4-HNE is nowadays considered as major bioactive marker of lipid peroxidation and a signaling molecule involved in regulation of several transcription factors sensible to stress such as nuclear factor erythroid 2-related factor 2 (Nrf2), activating protein-1 (AP-1), NF-κB, and peroxisome-proliferator-activated receptors (PPAR), in cell proliferation and/or differentiation, cell survival, autophagy, senescence, apoptosis, and necrosis (see below 4-HNE as signaling molecule).

Characteristics of various lipid peroxidation products as biomarkers have been reviewed on the basis of mechanisms and dynamics of their formation and metabolism and also on the methods of measurement, with an emphasis on the advantages and limitations [58].

2.3. Primary Lipid Peroxidation Product-Lipid Hydroperoxides

Hydroperoxides are produced during the propagation phase constituting the major primary product of lipid peroxidation process. The hydroperoxide group may be attached to various lipid structures, for example, free fatty acids, triacylglycerols, phospholipids, and sterols. Lipid hydroperoxide generation, turnover and effector action in biological systems have been reviewed [36]. In contrast to free radical, usually highly reactive and chemically unstable, at moderate reaction conditions, such as low temperature and absence of metal ions, lipid hydroperoxides are relatively more stable products. We found that lipid hydroperoxides in serum could be useful to predict the oxidative stress in tissues [59], and the levels of oxidative stress, including lipid peroxidation, increased throughout the day [60]. Once formed lipid hydroperoxides can be target of different reduction reactions, resulting in peroxidative damage inhibition or peroxidative damage induction.

Peroxidative Damage Inhibition. Hydroperoxides may decompose in vivo through two-electron reduction, which can inhibit the peroxidative damage. The enzymes mainly responsible for two-electron reduction of hydroperoxides are selenium-dependent glutathione peroxidases (GPx) and selenoprotein P (SeP). GPxs are known to catalyze the reduction of H2O2 or organic hydroperoxides to water or the corresponding alcohols, respectively, typically using glutathione (GSH) as reductant. Widely distributed in mammalian tissues GPx can be found in the cytosol, nuclei, and mitochondria [61, 62]. The presence of selenocysteine (in the catalytic centre of glutathione peroxidases) as the catalytic moiety was suggested to guarantee a fast reaction with the hydroperoxide and a fast reducibility by GSH [61]. SeP is the major selenoprotein in human plasma that reduced phospholipid hydroperoxide using glutathione or thioredoxin as cosubstrate. It protected plasma proteins against peroxynitrite-induced oxidation and nitration or low-density-lipoproteins (LDL) from peroxidation [62].

Peroxidative Damage Induction. Hydroperoxides may also decompose in vivo through one-electron reduction and take part in initiation/propagation steps [31, 36, 37], induce new lipid hydroperoxides, and feed the lipid peroxidation process; all these mechanisms can contribute to peroxidative damage induction/expansion. Lipid hydroperoxides can be converted to oxygen radicals intermediates such as lipid peroxyl radical () and/or alkoxyl () by redox cycling of transition metal (M), resulting in lipid hydroperoxide decomposition and the oxidized or reduced form of theses metal, respectively [63]. The lipid peroxyl and alkoxyl radicals can attack other lipids promoting the propagation of lipid peroxidation

Lipid hydroperoxides can also react with peroxynitrite (a short-lived oxidant species that is a potent inducer of cell death [64] and is generated in cells or tissues by the reaction of nitric oxide with superoxide radical) or hypochlorous acid (a high reactive species produced enzymatically by myeloperoxidase [65, 66], which utilizes hydrogen peroxide to convert chloride to hypochlorous acid at sites of inflammation) yielding singlet molecular oxygen [67, 68]. Singlet oxygen (molecular oxygen in its first excited singlet state 1Δg; 1O2)1 can react with amino acid, and proteins resulting in multiple effects including oxidation of side-chains, backbone fragmentation, dimerization/aggregation, unfolding or conformational changes, enzymatic inactivation, and alterations in cellular handling and turnover of proteins [69, 70].

Major substrates for lipid peroxidation are polyunsaturated fatty acids (PUFAs) [31, 36, 37], which are a family of lipids with two or more double bounds, that can be classified in omega-3 (n-3) and omega-6 (n-6) fatty acids according to the location of the last double bond relative to the terminal methyl end of the molecule. The predominant n-6 fatty acid is arachidonic acid (AA), which can be reduced (i) via enzymatic peroxidation to prostaglandins, leukotrienes, thromboxanes, and other cyclooxygenase, lipoxygenase or cytochrome P-450 derived products [4]; or (ii) via nonenzymatic peroxidation to MDA, 4-HNE, isoprostanes, and other lipid peroxidation end-products (more stables and toxic than hydroperoxides) through oxygen radical-dependent oxidative routes [49, 71]. The continued oxidation of fatty acid side-chains and released PUFAs, and the fragmentation of peroxides to produce aldehydes, eventually lead to loss of membrane integrity by alteration of its fluidity which finally triggers inactivation of membrane-bound proteins. Contrary to radicals that attack biomolecules located less than a few nanometres from its site of generation [22], the lipid peroxidation-derived aldehydes can easily diffuse across membranes and can covalently modify any protein in the cytoplasm and nucleus, far from their site of origin [72].

2.4. Secondary Lipid Peroxidation Products: MDA

MDA is an end-product generated by decomposition of arachidonic acid and larger PUFAs [49], through enzymatic or nonenzymatic processes (Figure 3). MDA production by enzymatic processes is well known but its biological functions and its possible dose-dependent dual role have not been studied although MDA is more chemically stable and membrane-permeable than ROS and less toxic than 4-HNE and methylglyoxal (MG) [49]. So far, only few papers have reported that MDA may act as signaling messenger and regulating gene expression: (i) very recent research indicated that MDA acted as a signaling messenger and regulated islet glucose-stimulated insulin secretion (GSIS) mainly through Wnt pathway. The moderately high MDA levels (5 and 10 μM) promoted islet GSIS, elevated ATP/ADP ratio and cytosolic Ca2+ level, and affected the gene expression and protein/activity production of the key regulators of GSIS [73]; (ii) in hepatic stellate cells, MDA induced collagen-gene expression by upregulating specificity protein-1 (Sp1) gene expression and Sp1 and Sp3 protein levels [74]. Both Sp1 and Sp3 can interact with and recruit a large number of proteins including the transcription initiation complex, histone modifying enzymes, and chromatin remodeling complexes, which strongly suggest that Sp1 and Sp3 are important transcription factors in the remodeling chromatin and the regulation of gene expression [75]. On the other hand, MDA production by nonenzymatic processes remains poorly understood despite their potential therapeutic value, because this MDA is believed to originate under stress conditions and has high capability of reaction with multiple biomolecules such as proteins or DNA that leads to the formation of adducts [76–78], and excessive MDA production have been associated with different pathological states [79–85] (see Table 1). Identifying in vivo MDA production and its role in biology is important as indicated by the extensive literature on the compound (over 15 800 articles in the PubMed database using the keyword “malondialdehyde lipid peroxidation” in December 2013).

Table 1: Common pathological processes linked to MDA and 4-HNE.

Figure 3: MDA formation and metabolism. MDA can be generated in vivo by decomposition of arachidonic acid (AA) and larger PUFAs as a side product by enzymatic processes during the biosynthesis of thromboxane A2 (TXA2) and 12-l-hydroxy-5,8,10-heptadecatrienoic acid (HHT) (blue pathway), or through nonenzymatic processes by bicyclic endoperoxides produced during lipid peroxidation (red pathway). One formed MDA can be enzymatically metabolized (green pathway). Key enzymes involved in the formation and metabolism of MDA: cyclooxygenases (1), prostacyclin hydroperoxidase (2), thromboxane synthase (3), aldehyde dehydrogenase (4), decarboxylase (5), acetyl CoA synthase (6), and tricarboxylic acid cycle (7).

MDA Production by Enzymatic Processes. MDA can be generated in vivo as a side product by enzymatic processes during the biosynthesis of thromboxane A2 (Figure 3) [86–90]. TXA2 is a biologically active metabolite of arachidonic acid formed by the action of the thromboxane A2 synthase, on prostaglandin endoperoxide or prostaglandin H2 (PGH2) [4, 91, 92]. PGH2 previously is generated by the actions of cyclooxygenases on AA [4, 91, 93].

MDA Production by Nonenzymatic Processes. A mixture of lipid hydroperoxides is formed during lipid peroxidation process. The peroxyl radical of the hydroperoxides with a cis-double bond homoallylic to the peroxyl group permits their facile cyclization by intramolecular radical addition to the double bond and the formation of a new radical. The intermediate free radicals formed after cyclization can cyclize again to form bicycle endoperoxides, structurally related to prostaglandins, and undergo cleavage to produce MDA. Through nonenzymatic oxygen radical-dependent reaction, AA is the main precursor of bicyclic endoperoxide, which then undergoes further reactions with or without the participation of other compounds to form MDA (Figure 3) [31, 49, 94, 95]. However, it should be possible that other eicosanoids that can also be generated by nonenzymatic oxygen radical-dependent reaction [96–99] may be precursor of bicyclic endoperoxide and MDA. Recent review has addressed the pathways for the nonenzymatic formation of MDA under specific conditions [100].

MDA Metabolism. Once formed MDA can be enzymatically metabolized or can react on cellular and tissular proteins or DNA to form adducts resulting in biomolecular damages. Early studies showed that a probable biochemical route for MDA metabolism involves its oxidation by mitochondrial aldehyde dehydrogenase followed by decarboxylation to produce acetaldehyde, which is oxidized by aldehyde dehydrogenase to acetate and further to CO2 and H2O (Figure 3) [49, 101, 102]. On the other hand, phosphoglucose isomerase is probably responsible for metabolizing cytoplasmic MDA to methylglyoxal (MG) and further to D-lactate by enzymes of the glyoxalase system by using GSH as a cofactor [103]. A portion of MDA is excreted in the urine as various enaminals (RNH-CH–CH-CHO) such as N-epsilon-(2-propenal)lysine, or N-2-(propenal) serine [49].

2.4.1. MDA Biomolecules Adducts

As a bifunctional electrophile aldehyde, MDA reactivity is pH-dependent, which exists as enolate ion (conjugate bases having a negative charge on oxygen with adjacent C–C double bond) with low reactivity at physiological pH. When pH decreases MDA exists as beta-hydroxyacrolein and its reactivity increases [49]. MDA’s high reactivity is mainly based on its electrophilicity making it strongly reactive toward nucleophiles, such as basic amino acid residues (i.e., lysine, histidine, or arginine). Initial reactions between MDA and free amino acids or protein generate Schiff-base adducts [49, 104, 175]. These adducts are also referred to as advanced lipid peroxidation end-products (ALEs). Acetaldehyde (product of MDA metabolism) under oxidative stress and in the presence of MDA further generates malondialdehyde acetaldehyde (MAA) adducts [157, 176]. MAA adducts are shown to be highly immunogenic [177–181]. MDA adducts are biologically important because they can participate in secondary deleterious reactions (e.g., crosslinking) by promoting intramolecular or intermolecular protein/DNA crosslinking that may induce profound alteration in the biochemical properties of biomolecules and accumulate during aging and in chronic diseases [72, 104, 182, 183]. Important proteins that can be modified by MDA adducts are as follows: (i) eElongation factor 2 (eEF2) catalyzes the movement of the ribosome along the mRNA in protein synthesis. MDA adducts with eEF2 could contribute to decline of protein synthesis, secondary to LP increase (see below—cumene hydroperoxide-induced lipid peroxidation); (ii) factor H (FH) is the main regulator of the alternative pathway in plasma that tightly controls the activation of complement to prevent attack against host cells. MDA adducts with FH can block both the uptake of MDA-modified proteins by macrophages and MDA-induced proinflammatory effects in vivo in mice [184]; MDA adducts or MAA adducts can promote binding of complement; (iii) anaphylatoxin C3a (proinflammatory complement components) with oxidatively modified low-density lipoproteins (Ox-LDL) and contributes to inflammatory processes involving activation of the complement system in atherosclerosis [185]; and (iv) protein kinase C (PKC) is known to play a major role in intracellular signal transduction affecting such processes as proliferation, differentiation, migration, inflammation, and cytoskeletal organization. BSA-MAA induces the activation of a specific isoform of PKC, PKC-α, in hepatic stellate cells (HSCs) and induces the increased secretion of urokinase-type plasminogen activator, a key component of the plasmin-generating system, thereby contributing to the progression of hepatic fibrosis [186]. A recent review shows a list of up to thirty-three proteins known to be modified by MDA and including enzymatic proteins, carrier proteins, cytoskeletal proteins, and mitochondrial and antioxidant proteins [76].

It has also been proposed that MDA could react physiologically with several nucleosides (deoxy-guanosine and cytidine) to form adducts to deoxyguanosine and deoxyadenosine, and the major product resulting is a pyrimidopurinone called pyrimido[1,2-a]purin-10(3H-)one (M1G or M1dG) [122, 123, 187, 188]. MDA is an important contributor to DNA damage and mutation [122, 124]. The main route for repair of M1dG residues in genomic DNA appears to be the nucleotide excision repair (NER) pathway [188, 189]. In the absence of repair, MDA-DNA adducts may lead to mutations (point and frameshift) [124], strand breaks [122, 190], cell cycle arrest [191], and induction of apoptosis [192]. M1dG is oxidized to 6-oxo-M1dG in rats and that xanthine oxidase (XO) and aldehyde oxidase (AO) are the likely enzymes responsible [193]. This MDA-induced DNA alteration may contribute significantly to cancer and other genetic diseases. Hypermethylated in cancer 1 (HIC1) is a tumor suppressor gene that cooperates with p53 to suppress cancer development. New funding has shown that highest HIC1 methylation levels in tobacco smokers were significantly correlated with oxidative DNA adducts M1dG [125]. Research also suggests that persistent M1dG adducts in mitochondrial DNA hinder the transcription of mitochondrial genes [194]. Dietary intake of certain antioxidants such as vitamins was associated with reduced levels of markers of DNA oxidation (M1dG and 8-oxodG) measured in peripheral white blood cells of healthy subjects, which could contribute to the protective role of vitamins on cancer risk [195].

2.5. Secondary Lipid Peroxidation Products: 4-HNE

4-Hydroxynonenal (4-HNE), α, β-unsaturated electrophilic compounds, is the major type of 4-hydroxyalkenals end-product, generated by decomposition of arachidonic acid and larger PUFAs, through enzymatic or nonenzymatic processes [49]. 4-HNE is an extraordinarily reactive compound containing three functional groups: (i) C=C double bond that can be target to Michael additions to thiol, reduction or epoxidation, (ii) carbonyl group which can yield acetal/thio acetal or can be target to Schiff-base formation, oxidation, or reduction, and (iii) hydroxyl group which can be oxidized to a ketone [56].

4-HNE is the most intensively studied lipid peroxidation end-product, in relation not only to its physiological and protective function as signaling molecule stimulating gene expression, but also to its cytotoxic role inhibiting gene expression and promoting the development and progression of different pathological states. In the last three years, excellent reviews have been published summarizing both signaling and cytotoxic effects of this molecule in biology, for example, overview of mechanisms of 4-HNE formation and most common methods for detecting and analyzing 4-HNE and its protein adducts [196]. Review focuses on membrane proteins affected by lipid peroxidation-derived aldehydes, under physiological and pathological conditions [131]. Jaganjac and Co-workers have described the role of 4-HNE as second messengers of free radicals that act both as signaling molecules and as cytotoxic products of lipid peroxidation involvement in the pathogenesis of diabetes mellitus (DM) [151]. Chapple and Co-workers summarized the production, metabolism and consequences of 4-HNE synthesis within vascular endothelial, smooth muscle cells and targeted signaling within vasculature [142]. Review focuses on the role of 4-HNE and Ox-PLs affecting cell signaling pathways and endothelial barrier dysfunction through modulation of the activities of proteins/enzymes by Michael adducts formation, enhancing the level of protein tyrosine phosphorylation of the target proteins, and by reorganization of cytoskeletal, focal adhesion, and adherens junction proteins [197]. An overview of molecular mechanisms responsible for the overall chemopreventive effects of sulforaphane (SFN), focusing on the role of 4-HNE in these mechanisms, which may also contribute to its selective cytotoxicity to cancer cells [198]. Perluigi and Co-workers summarized the role of lipid peroxidation, particularly of 4-HNE-induced protein modification, in neurodegenerative diseases. In this review, the authors also discuss the hypothesis that altered energy metabolism, reduced antioxidant defense, and mitochondrial dysfunction are characteristic hallmarks of neurodegenerative [170]. Zimniak described the effects of 4-HNE and other endogenous electrophiles on longevity, and its possible molecular mechanisms. The role of electrophiles is discussed, both as destabilizing factors and as signals that induce protective responses [199]. Reed showed the relationship between lipid peroxidation/4-HNE and neurodegenerative diseases. It also demonstrates how findings in current research support the common themes of altered energy metabolism and mitochondrial dysfunction in neurodegenerative disorders [171]. Fritz and Petersen summarized the generation of reactive aldehydes via lipid peroxidation resulting in protein carbonylation, and pathophysiologic factors associated with 4-HNE-protein modification. Additionally, an overview of in vitro and in vivo model systems used to study the physiologic impact of protein carbonylation, and an update of the methods commonly used in characterizing protein modification by reactive aldehydes [200]. Butterfield and Co-workers showed that several important irreversible protein modifications including protein nitration and 4-HNE modification, both which have been extensively investigated in research on the progression of Alzheimer's disease (AD) [201]. Balogh and Atkins described the cellular effects of 4-HNE, followed by a review of its GST-catalyzed detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha-class GSTs. Additionally, a summary of the literature that examines the interplay between GSTs and 4-HNE in model systems relevant to oxidative stress is also discussed to demonstrate the magnitude of importance of GSTs in the overall detoxification scheme [202]. Like MDA, 4-HNE has high capability of reaction with multiple biomolecules such as proteins or DNA that lead to the formation of adducts [49].

4-HNE Production by Enzymatic Processes. 4-HNE is a lipid peroxidation end-product of enzymatic transformation of n-6 PUFAs (AA, linoleic acid, and other) by 15-lipoxygenases (15-LOX). Two different 15-LOX exist, (i) 15-LOX-1 (reticulocyte type) expressed in reticulocytes, eosinophils, and macrophages; (ii) and 15-LOX-2 (epidermis type) expressed in skin, cornea, prostate, lung, and esophagus [203–205]. Mice do not express 15-LOX and only express the leukocyte-derived 12-LOX. In plant enzymatic route to 4-HNE includes lipoxygenase (LOX), -hydroperoxide lyase (HPL), alkenal oxygenase (AKO), and peroxygenases (Figure 4) [206]. The main precursors of 4-HNE in human are 13-hydroperoxyoctadecadienoic acid (13-HPODE) produced by the oxidation of linoleic acid by 15-LOX-1 [207] and 15- hydroperoxyeicosatetraenoic acids (15-HPETE) produced by the oxidation of AA by 15-LOX-2 [208]. These compounds are short lived and are catabolised into various families of more stable compounds such as 15-HETEs, lipoxins, and leukotrienes [4]. 15-HPETE is associated with anti-inflammatory and proapoptotic functions (the release of cytochrome c, activation of caspase-3 and 8, PARP, and Bid cleavage) and DNA fragmentation [209, 210].

Figure 4: Enzymatic production of 4-HNE and metabolism. In plant enzymatic route to 4-HNE includes lipoxygenase (LOX), -hydroperoxide lyase (HPL), alkenal oxygenase (AKO), and peroxygenases. 4-HNE metabolism may lead to the formation of corresponding alcohol 1,4-dihydroxy-2-nonene (DHN), corresponding acid 4-hydroxy-2-nonenoic acid (HNA), and HNE–glutathione conjugate products. 4-HNE conjugation with glutathione s-transferase (GSH) produce glutathionyl-HNE (GS-HNE) followed by NADH-dependent alcohol dehydrogenase (ADH-)catalysed reduction to glutathionyl-DNH (GS-DNH) and/or aldehyde dehydrogenase (ALDH-)catalysed oxidation to glutathionyl-HNA (GS-HNA). 4-HNE is metabolized by ALDH yielding HNA, which is metabolized by cytochrome P450 (CYP) to form 9-hydroxy-HNA (9-OH-HNA). 4-HNE may be also metabolized by ADH to produce DNH.

4-HNE Production by Nonenzymatic Processes. 4-HNE can be formed through several nonenzymatic oxygen radical-dependent routes involving the formation of hydroperoxides, alkoxyl radicals, epoxides, and fatty acyl crosslinking reactions. Spickett C [196] recently reviewed the mechanisms of formation of 4-HNE during lipid peroxidation and showed that the main processes leading to 4-HNE are likely beta-cleavage reaction of lipid alkoxy-radicals, which can be summarized into five generic mechanisms: (i) reduction of the hydroperoxide to a lipid alkoxy radical by transition metal ions, such as Fe2+ followed by b-scission; (ii) protonation of the lipid hydroperoxide yields an acidified lipid hydroperoxide that undergoes Hock rearrangement of a C–C to C–O bond followed by hydrolysis and Hock cleavage; (iii) the lipid peroxyl radical of the hydroperoxides permits their facile cyclization to dioxetane and ending with dioxetane cleavage; (iv) free radical attack to ω-6 PUFA on bis-allyl site yielding a free radical intermediate, that further reacts with molecular oxygen to generate hydroperoxide derivatives such as 13-HPODE or 15-HPETE. The abstraction of an allylic hydrogen of their structure produce another radical intermediate that after oxygenation step forms the corresponding dihydroperoxyde derivative (unstable), which after Hock rearrangement and cleavage produces 4-hydroperoxy-2E-nonenal (4S-HPNE), immediate precursor of HNE; and (v) the oxidation products generated after reaction of linoleate-derived hydroperoxy epoxide (13-Hp-Epo-Acid) with Fe+2 yields an alkolxyl radical, which undergo to di-epoxy-carbinyl radical and after beta-scission yield different aldehydes compounds including 4-HNE (Figure 5).

Figure 5: Nonenzymatic 4-HNE production. Initial abstraction of bisallylic hydrogen of lipoic acid (LA) produces fatty radicals. 4-HNE formation starting with 9- and 13-hydroperoxyoctadecadienoate (HPODE) (red and blue pathways, resp.). 4-HNE is generated by beta-scission of a hydroxyalkoxy radical that is produced after cyclization of alkoxy radical in the presence of transition metal ions and two molecules of oxygen; this reaction involves hydrogen abstraction (1). Peroxy radical cyclizes to form a dioxetane which is oxygenated to peroxy-dioxetane that is fragmented and after two hydrogen abstractions produce 4-HNE (2). Hydroperoxyl radical is oxygenated to dioxetane that is further fragmented to produce 4-hydroperoxy-2E-nonenal (4-HPNE), an immediate precursor of 4-HNE (3). Bicyclic endoperoxides react with reduced form of transition metal, such as iron (Fe2+) to produce alkoxyl radicals which after reaction with oxygen (O2), hydrogen abstraction (H+), and fragmentation produce 4-HNE (4). Alkoxyl radical after cyclization, oxygenation, hydrogen abstraction, oxidation of transition metal, hydrolysis, and rearrangement yields 4-HNE (5). With arachidonic acid, 11- and 15- hydroperoxyeicosatetraenoic acids (HPETE) are the precursors to form 4-HNE via the analogous mechanisms.

Once formed 4-HNE, and depending of cell type and cellular metabolic circumstances can promote cell survival or death. Cells expressing differentiated functions representative for the in vivo situation react more sensitively to 4-HNE than cell lines. The different response with respect to the endpoints of genotoxicity probably depends on the different metabolizing capacities and thus the action of different metabolites of 4-HNE [211]. 4-HNE can be enzymatically metabolized at physiological level and cells can survive; 4-HNE can play an important role as signaling molecule stimulating gene expression (mainly Nrf2) with protective functions that can enhance cellular antioxidant capacity and exert adaptive response when 4-HNE level is low; under this circumstances cells can survive; 4-HNE can promote organelle and protein damage leading to induction of autophagy, senescence, or cell cycle arrest at 4-HNE medium level and cells can subsist; and finally 4-HNE induces apoptosis or necrosis programmed cell death at 4-HNE high or very high level, respectively, and cells die. These processes eventually lead to molecular cell damage which may facilitate development of various pathological states. High levels of 4-HNE can also react with proteins and/or DNA to form adducts resulting in a variety of cytotoxic and genotoxic consequences (Figure 6).

Figure 6: 4-HNE promotes cell survival or induces cell death. Depending on cell type, damage/repair capacities and cellular metabolic circumstances 4-HNE can promote cell survival or induce cell death. 4-HNE at physiological levels is enzymatically metabolized and at low levels plays an important role as signaling molecule stimulating gene expression, enhance cellular antioxidant capacity and exert adaptive response; at medium levels organelle and protein damage lead to induction of autophagy, senescence, or cell cycle arrest and at high or very high levels promote adducts formation and apoptosis or necrosis cell death, respectively.

4-HNE Metabolism. The main goal of the rapid intracellular metabolism of 4-HNE in mammalian cells is to protect proteins from modification by aldehydic lipid peroxidation products [212]. The biochemical routes of 4-HNE metabolism that lead to the formation of corresponding alcohol 1,4-dihydroxy-2-nonene (DHN), corresponding acid 4-hydroxy-2-nonenoic acid (HNA), and HNE-glutathione conjugate products can be summarized according to stress levels: (i) under physiological or low stress levels the major 4-HNE detoxification step is conjugation with GSH to yield glutathionyl-HNE (GS-HNE) or glutathionyl-lactone (GS-)lactone (cyclic ester 4-HNE- form) followed by NADH-dependent alcohol dehydrogenase (ADH-)catalysed reduction to glutathionyl-DNH (GS-DNH) and/or aldehyde dehydrogenase (ALDH-)catalysed oxidation to glutathionyl-HNA (GS-HNA); (ii) at moderate stress levels, 4-HNE undergoes aldehyde dehydrogenase (ALDH-)catalysed oxidation yielding HNA, that may be further metabolized in mitochondria through beta-oxidation by cytochrome P450 to form 9-hydroxy-HNA; and (iii) at high stress levels, 4-HNE is metabolized by ADH (that belongs to the aldo-keto reductase (AKR) superfamily) to produce DNH [131, 196, 202, 212, 213] (Figure 4). By disrupting the Gsta4 gene that encodes the alpha class glutathione s-transferase (GST) isozyme GSTA4-4 in mice showed that GSTA4-4 plays a major role in protecting cells from the toxic effects of oxidant chemicals by attenuating the accumulation of 4-HNE [214]. Overexpression and inhibition of ALDH activity reduce and increase, respectively, the 4-HNE toxicity and 4-HNE-protein adducts levels in cell culture [215, 216].

2.5.1. 4-HNE as Signaling Molecule

At moderate concentration, when the basal level of antioxidant enzymes cannot be sufficient to neutralize 4-HNE, cells can survive due to 4-HNE may regulate several transcription factors sensible to stress such as nuclear factor erythroid 2-related factor 2 (Nrf2), activating protein-1 (AP-1), NF-κB, and peroxisome-proliferator-activated receptors (PPAR). It also activates stress response pathways such as mitogen-activated protein kinases (MAPK), EGFR/Akt pathways, and protein kinase C. Different labs demonstrated the 4-HNE-dependent induction of Nrf2, a primary sensor and oxidative stress regulator [217–221]. Also administration of the Nrf2-ARE activators protect from 4-HNE toxicity [222]. Under physiological conditions, Nrf2 is sequestered in the cytoplasm by the repressor protein Keap1, but in response to oxidant stimuli Nrf2 is activated and translocated into the nucleus where mediate the transcription of antioxidant/cytoprotective genes by binding to the antioxidant-response element (ARE) within DNA [223]. The Nrf2-ARE pathway has essential role in different pathological states such as neurodegenerative diseases [223], cancer [224], diabetes [225], and infectious disease [226]. The main genes regulated by 4-HNE- induced Nrf2-ARE pathway are as follows: (i) HO-1, an antioxidant protein that catalyzes the degradation of heme to biliverdin, which is then degraded to bilirubin; both biliverdin and bilirubin have antioxidant properties [227]; 4-HNE can upregulate HO-1 [217, 220, 221, 228–230]; (ii) thioredoxin (Trx) and thioredoxin reductase (TrxR); Trx is a small (13 kDa) antioxidant ubiquitous protein with two redox-active cysteine residues (-Cys-Gly-Pro-Cys-) in its active center; oxidized Trx is reduced back to the active form of Trx by Trx reductase (TrxR) in the presence of NADPH [231]; 4-HNE can upregulate Trx/TrxR [220, 221, 232]; (iii) glutamate cystein ligase (GCL) is a major determinant enzyme in GSH synthesis [233, 234]. 4-HNE can upregulate GCL [235–239].

Involvement of AP-1 transcription factor in 4-HNE-induced cell signaling has been demonstrated by several studies which showed an AP-1 upregulation by 4-HNE [240–243]. Activation of AP-1 binding may lead to the 4-HNE-induced increase in GSH content [239]. AP-1 is a dimer consisting of basic region-leucine zipper proteins from the Jun and Fos subfamilies. AP-1 transcription factors control cell proliferation, survival, and death. Growth factors, cytokines, cellular stress, and many other stimuli activate AP-1 [244, 245].

NF-κB is a dimeric transcription factor that regulates diverse biological processes, including immune responses, inflammation, cell proliferation, and apoptosis. The NF-κB protein complex is retained in an inactive state in the cytoplasm by binding to inhibitory proteins IκBs family [246]. Various cellular stimuli, such as oxidative stress, IκBs are phosphorylated, making them susceptible to degradation by the ubiquitin-proteasome system. This results in nuclear translocation of NF-κB complex where it can bind to various promoter areas of its target genes and induce gene transcription of the corresponding genes [246, 247], most of which are implicated in the regulation of inflammation. 4-HNE can activate or inhibit NF-κB depending on the type of cells used. For example, 4-HNE inhibited the activity of NF-κB in hepatocytes [165], cortical neurons [248], ARPE-19 human retinal pigment epithelial cells [249], Kupffer cells [250], human aortic endothelial cells [251], human colorectal carcinoma, and lung carcinoma cell [252]. On the contrary, 4-HNE induced activity of NF-κB in macrophages [253], vascular smooth muscle cells [254], PC12 cells [255], optic nerve head astrocytes [256], human osteoarthritic chondrocytes [257], human fibroblasts [258], and human monocytic lineage cells [259].

PPARs comprise three subtypes (PPARα, β/δ, and γ) to form a nuclear receptor superfamily. PPARs act as key transcriptional regulators of lipid metabolism, mitochondrial biogenesis, and antioxidant defense [260, 261]. PPARs interaction/modulation with 4-HNE has been reviewed [262]. 4-HNE increased PPAR-γ gene expression and accelerated adiponectin protein degradation in adipocytes [263]; expression of PPAR-γ was induced in HL-60 and U937 cells by 4-HNE treatment [264], whereas in the colon cancer cell (CaCo-2) PPARγ protein expression was not induced after 4-HNE treatment [265]; 4-HNE increased PPARγ2 expression in C2C12 cells [266]. PPAR-β/δ is activated by 4-HNE in 3T3-L1 preadipocytes cells [267]. 4-HNE activates PPAR-δ and amplifies insulin secretion in INS-1E β-cells [152].

MAP kinases family can be activated in response to diverse stimuli such as oxidative stress, lipopolysaccharides, inflammatory cytokines, growth factors, or endoplasmic reticulum (ER) stress and are involved in several cellular responses like cell proliferation and/or differentiation, inflammation, proteasomal-mediated protein degradation, and apoptosis. Members of the major mitogen-activated protein kinase (MAPK) subfamilies are the extracellular signal-regulated kinase (ERK), p38, and Jun N-terminal kinase (JNK) subfamilies. The mechanism by which MAPK signaling cascades are activated by 4-HNE is not well known. For example, activation of different MAPK under various stimuli can affect both apoptotic and prosurvival signaling. In corneal epithelial cells, 4-HNE caused a time-dependent induction of HO-1 mRNA and protein via modification and activation of Erk1/2, JNK and p38 MAP kinases, as well as phosphoinositide-3-kinase (PI3)/Akt. Inhibition of p38 blocked 4-HNE-induced HO-1 expression; inhibition of Erk1/2 and, to a lesser extent, JNK and PI3 K/Akt suppressed 4-HNE-induced HO-1 [268]. 4-HNE also stimulated Erk1/2, JNK, p38, and PI3 kinase in keratinocyte, and the inhibitors of these enzymes suppressed 4-HNE-induced expression of HO-1 [269]. In PC12 cells, 4-HNE treatment induced ERK, JNK, and p38 MAPK activation as well as induced the expression of HO-1. Addition of p38 MAPK specific inhibitor SB203580 attenuated HO-1 upregulation; these results indicate that 4-HNE-induced transient p38 MAPK activation may serve as an upstream negative regulator of ER stress and confer adaptive cytoprotection against 4-HNE-mediated cell injury [228]. In rat liver epithelial RL34 cells, 4-HNE upregulates the cyclooxygenase-2 (COX-2, which plays a key role in conversion of free arachidonic acid to PGs) expression by the stabilization of COX-2 mRNA via activation of the p38 MAPK pathway [270]. In human hepatic stellate cells (hHSC), 4-HNE forms adducts with JNK and this event leads to JNK nuclear translocation and activation as well as to c-jun and AP-1 induction [271]. In human bronchial epithelial cells, 4-HNE downmodulates the protein-tyrosine phosphatase SH2 domain containing phosphatase-1 (SHP-1) which negatively regulates JNK activity [272]. We can also see the protective effects of MAPK activation via GSH induction because the activation of the ERK pathway is involved in GCL (the rate-limiting enzyme in de novo glutathione (GSH) synthesis) regulation in rat cells [273] while the JNK pathways appear to be involved in human HBE-1 cells [274].

In human monocytes, 4-HNE was shown to significantly inhibit p38 and ERK activity, which resulted in inhibition of TNF and interleukin-1beta production in response to LPS. The data suggest that 4-HNE, at nontoxic concentrations, has anti-inflammatory properties [275]. In human osteoarthritic osteoblasts, 4-HNE also showed a significant (approximately 70%) decrease of TNF-α-induced IL-6 mRNA expression via the NF-κB signaling pathway. However, only p38 MAPK and JNK1/2 were activated, but not ERK1/2 [276], while 4-HNE also induced COX-2 expression and prostaglandin E2 (PGE2) release [257, 276].

On the other hand, 4-HNE mediated depletion of intracellular thiols, protein tyrosine phosphorylation, MAPK (JNK, ERK, and p38) activation, and modulates integrin resulting in reorganization of cytoskeletal, focal adhesion proteins, and barrier dysfunction in lung microvascular endothelial cells [277]. Results suggest that activation and phosphorylation of MAP kinases (JNK, ERK, and p38) play an important role in 4-HNE mediated toxicity and cell death in mouse embryonic fibroblasts (MEF), and absence of GSTA4–4 potentiates the cytotoxic effects of 4-HNE. The increase of apoptosis in Gsta4 null MEF by 4-HNE was associated with the enhanced accumulation of 4-HNE-protein adducts, DNA damage, and the activation of caspases-3, -8, and -9 [214]. 4-HNE upregulates and phosphorylates cytosolic phospholipase A-2 (cPLA-2) in cultured microglial cell line (Ra2) via the ERK and p38 MAPK pathways [278]. cPLA is a proinflammatory enzyme that stimulate AA- release by hydrolyzes glycerophospholipids with AA in the sn-2 position.

Matrix metalloproteinases (MMPs) constitute a large group of endoproteases that are not only able to cleave all protein components of the extracellular matrix but also to activate or inactivate many other signaling molecules, such as receptors, adhesion molecules, and growth factors [279]. 4-HNE induced MMP-9 production in macrophages [280] and MMP-2 in vascular smooth muscle cells (VSMC) [281] via activation of ERK and p38 MAPK pathways, consequently leading to plaque instability in atherosclerosis. 4-HNE also enhances MMP-2 production in VSMC via mitochondrial ROS-mediated activation of the Akt/NF-kappaB signaling pathways [254]. In osteoarthritic (OA) synovial cells, 4-HNE induced MMP-13 mainly through activation of p38 MAPK [282].

Akt (a.k.a protein kinase B or PKB) comprises three closely related isoforms Akt1, Akt2, and Akt3 (or PKBα/β/γ resp.), which play a role in the regulation of cell proliferation, survival, and metabolism. Dysregulation of Akt leads to diseases such as cancer, diabetes, and cardiovascular and neurological diseases [283]. Under conditions of enhanced oxidative stress, a major cellular response is the activation of the Akt pathway that involves the oxidation and subsequent inactivation of PTEN (phosphatase and tensin homolog deleted on chromosome 10), a tumor suppressor and primary regulator of Akt [284]. Recent studies have also demonstrated that activation of PI3 K/Akt signaling by 4-HNE occurs via modification and inhibition of PTEN, a regulatory protein that suppresses Akt2 activity, which is selectively phosphorylated by 4-HNE in both cellular human hepatocellular carcinoma cell line (HepG2) [285] and animal models (ethanol-fed mice) [286]. In HepG2 cells, 4-HNE inhibits H2O2-mediated activation of the Akt pathway in leading to phosphorylation of Akt1 but not Akt2, decreased cell proliferation, and decreased expression of cyclin D1 [287]. In retinal pigment epithelial (RPE) cells, at lower concentrations 4-HNE triggered phosphorylation of epidermal growth factor receptor (EGFR) and activation of its downstream signaling components ERK1/2 and Akt; this led to protective mechanism against oxidative stress [288]. Akt- induced activity by 4-HNE promotes cell survival through induction of HO-1 mRNA and protein in corneal epithelial cells [268], and in keratinocyte [269]. The inhibitors of Akt suppressed 4-HNE-induced expression of HO-1.

Protein kinases C (PKCs) are a family of multifunctional enzymes that play crucial roles in the transduction of many cellular signals such as control of cell proliferation, survival, and transformation by phosphorylating various targets. The PKC family consists of three different groups: conventional (α, β1, β2, and γ), novel (δ, ε, η, and θ), and atypical (ζ and λ/τ). Conventional and novel PKC isoforms are lipid-sensitive enzymes and calcium-dependent and are usually activated by growth factors through stimulation of phospholipase C (PLC) which hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) to generate inositol triphosphate (IP3) and DAG [6, 289]. Cells can express more than one PKC isoform, and individual PKCs can mediate different biological processes. For example, in human promyelocytic leukemia (HL-60) cells [290–292] and rat neutrophils [293] 4-HNE induced a significant increase of PLC activity, which should result in an increased production of IP3 and DAG, known to stimulate PKC [289]. Phagocytes, such as granulocytes and monocytes/macrophages which engulf microbial intruders and effectively kill and eradicate the foreign bodies, contain a membrane-associated NADPH oxidase that produces superoxide leading to other ROS with microbicidal, tumoricidal, and inflammatory activities [294]. In RAW 264.7 mouse macrophage cells, 4-HNE exhibited a concentration-dependent inhibition of ROS by adduction to PKC, a protein vital in the assembly and activation of NADPH oxidase [295]. In rat hepatocyte PKC- isoforms activity is differentially regulated by concentrations 4-HNE. For example, PKC-α activity was decreased in a dose-dependent manner by all concentrations of 4-HNE, while low concentrations of 4-HNE increased PKC βI and, to a much greater extent, PKC βII activities. By contrast, they were unaffected or even inhibited by higher concentrations of 4-HNE. This PKC-dependent- 4-HNE regulation could be involved in the traffic of secretory glycoproteins [296]. In NT2 neurons, low 4-HNE concentrations (similar to concentrations detected in AD brain tissue) induced a 2–6 fold increase of intracellular amyloid β-protein (Aβ) production that was concomitant with selective activation of βI and βII PKC isoforms [297, 298]. In macrophages, a marked and early upregulation of monocyte chemoattractant protein 1 (MCP-1) release occurs in response to low 4-HNE concentrations, most likely through of the increase in the activity of PKC-βI and βII classic isoforms, while the activation of PKC-δ appeared to be involved in LPS-stimulated cells [299]. Treatment of macrophages with 4-HNE, cell-permeable esters of glutathionyl-4-hydroxynonenal (GS-HNE) and glutathionyl-1,4-dihydroxynonane (GS-DHN) activated NF-κB and PLC/PKC. Aldolase reductase catalyzes the reduction of GS-HNE to GS-DHN. AR inhibition/ablation prevented PLC, PKC, and IKKalpha/beta, and NF-κB activation caused by 4-HNE and GS-HNE, but not by GS-DHN, suggests a novel role for a reduced glutathione-lipid aldehyde conjugate (such as GS-DHN) as an obligatory mediator of ROS-induced cytotoxicity [300].

2.5.2. Effect of 4-HNE on Autophagy

One of the most important processes for maintaining normal metabolic and redox signaling, through degradation of damaged proteins and organelles, is autophagy-lysosomal pathway [301]. 4-HNE can promote protein-adducts leading to protein damage and to induction of autophagy-lysosomal pathway [302], a process that is increased by treatment with an autophagy stimulator, rapamycin. If autophagy is blocked with a PI3 K inhibitor, 3-methyladenine, apoptotic cell death occurs [301, 302]. Several mechanisms by which 4-HNE induces autophagy have been reported. For example, 4-HNE promotes the formation of protein adducts that accumulate in the endoplasmic reticulum (ER) and led to autophagy in rat aortic smooth muscle cells, through selective activation of the PKR-like ER kinase (PERK) pathway accompanied by JNK activation, the upregulation of the HO-1, increased microtubule-associated protein 1 light chain 3 (LC3) formation, and maintenance of cell viability under conditions of excessive 4-HNE-protein adducts accumulation [303]. In differentiated SH-SY5Y neuroblastoma cells, glucose-dependent autophagy serves as a protective mechanism in response to 4-HNE because low 4-HNE-concentrations increased autophagy and induced concentration dependent CASP3/caspase-3 activation and cell death. Additionally inhibition of glucose metabolism by 2-deoxyglucose and glycolysis by koningic acid, a GAPDH, inhibitor, led to autophagy inhibition and increased CASP3 activation and cell death [304]. On the contrary, phagocytosis of 4-HNE- and MDA-modified photoreceptor outer segments (POS) induced a marked reduction of autophagic activity by 40% in retinal pigment epithelium (RPE) cells, which may contribute to RPE cell dysfunction and degeneration. In contrast, unmodified POS had no significant effect on autophagy [305].

2.5.3. Effect of 4-HNE on Senescence

Cellular senescence, defined as arrest during the cell cycle (G0), is involved in the complex process of the biological aging of tissues, organs, and organisms. Senescence is driven by many factors including oxidative stress, the DNA damage/repair response, inflammation, mitogenic signals, and telomere shortening. Telomeres are considered a “biological clock” of the cell and are shortened by each cell division until a critical length is reached and dysfunction ensues. Rapid telomere shortening may indicate a very high cellular activity. DNA-repair pathways are then recruited and cells enter senescence, losing their capacity to proliferate. In addition to cell division, factors causing telomere shortening include DNA damage, inflammation, and oxidative stress [306]. Activation of a DNA damage response including formation of DNA damage foci containing activated H2A.X (γ-histone 2A.X) at either uncapped telomeres or persistent DNA strand breaks is the major trigger of cell senescence. γH2AX is a sensitive marker of DNA damage, particularly induction of DNA double-strand breaks [307]. The length of telomeres depends on the telomerase activity and the catalytic subunit of telomerase (hTERT) which is strongly upregulated in most human cancers [308], and the major consequence of the reactivation of telomerase activity is that tumor cells escape from senescence. The expression of c-myc (an activator), mad-1 (a repressor) and sp-1 (an activator/repressor), which have been shown to activate hTERT transcription. The formation of 4-HNE-proteins adducts in general increased as a function of age [309]. Quantitative evaluation showed that the majority of senescent hepatocytes (as measured by γ-H2A.X) were also positive for 4-HNE [310, 311]. 4-HNE can induce premature senescence by a direct suppression of telomerase activity affecting the expression of hTERT. In endothelial cells (EC) isolated and cultured from arterial segments of patients with severe coronary artery disease, chronic treatment with an antioxidant (that significantly decreased the levels of lipid peroxidation, that is, 4-HNE expression) N-acetyl-cystein, NAC, significantly delayed cellular senescence via decrease of DNA damage marker (γH2AX), decrease of nuclear p53, and increase in hTERT activity [312]. In three human leukemic cell lines (HL-60, U937, and ML-1) [313] and in colon cancer cells (Caco-2 and HT-29) [314], telomerase activity and hTERT expression were downregulated by 4-HNE, as a consequence of downregulation of c-myc mRNA expression and c-Myc DNA binding activity as well as upregulation of mad-1 mRNA expression and Mad-1 DNA binding activity. On the other hand, 4-HNE may induce cellular senescence through activation of critical cell cycle sentinels that mediate this process, such as the tumor suppressor proteins p53 (see below), which is well known to play a central role in senescence [315–320]. p53 protects cells of oxidative stress and promotes DNA repair. However, when in the cells the extent of damage overwhelms repair capacities, p53 induces cell death [315–319]. All these data thus confirmed a cell-specific association between senescence and 4-HNE.

2.5.4. Effect of 4-HNE on Cell Cycle and Proliferation

In cell cycle the transition of different phases is driven by several phase-specific cyclin-CDK (cyclin-dependent kinase) complexes which previously have been activated. In response to mitogens, cyclin D is activated and phosphorylate retinoblastoma protein (RB) which leads to activation of E2F proteins and the expression of E2F-responsive genes inducing cells to reenter the cell cycle from quiescence called G0, to G1. Activation of E2F leads to the transcription of cyclin E for transition from G1 to S phase. Subsequent expression of cyclin A leads to transition of S to G2 and cyclin B leads G2 to M phases [321, 322]. The promitotic factor Cdc25 stimulates cell cycle progression through the activation of cyclin A-Cdk1, cyclin B-Cdk1, and cyclin E-Cdk2 for entry into M phase by removing the inhibitory phosphorylation on Cdk1 and Cdk2. On the contrary, the anti-mitotic factor (p21, p27, p57) inhibit cell cycle progression through inhibition of cyclin A–Cdk1, cyclin B–Cdk1, cyclin E–Cdk2 and cyclin D–Cdk4/6 [321–323]. In response to 4-HNE, the expression of key components of cell cycle can be modulated and cells are arrested at G1 or G2. Several studies showed that in general 4-HNE may induce cell cycle arrest in malignant cell and inhibition or decrease of cell proliferation. For example, treatment of HL-60 cells with 4-HNE (1 μM) causes a p53-independent increase of p21 expression, RB dephosphorylation, progressive reduction in the amount of free E2F bound to DNA, and a relative increase in E2F complexes at higher molecular weights with repressive activity decrease of E2F complexes [324], and decrease of cyclin D1, cyclin D2, and cyclin A [325]. In human erythroleukemia cells (K562), 4-HNE treatment increased p53 and p21 expression and decreased expression of cyclin D2. The additional decrease of A- and B-cyclin suggests that the S- and G2-phase were also retarded contributing to the overall slowdown of the cycle [326]. In human breast cancer cells (MCF7) the increase in endogenous levels of 4-HNE caused by treatment with conjugated linoleic acid (CLA) resulted in the inhibition of cell proliferation through a p53-dependent mechanism [327]. In human osteosarcoma cells (HOS), 4-HNE treatment declined gradually the proportion of cells in mitosis, inhibited proliferation and differentiation, and increased apoptosis [328]. In malignant cells like hepatome cells, with a below-normal content of PUFAs and very high expression of aldehyde dehydrogenase-3 (ADH3) which metabolize 4-HNE to DNH, the inhibitory effects of 4-HNE on cell proliferation are lower, but the inhibition of ADH3 resulted in an increase in the quantity of aldehyde in the cells and inhibit cell proliferation through the MAPK pathway by reduction of pRaf-1 and pERK1,2 [329, 330]. Moreover, 4-HNE has also antiproliferative/differentiative effect mainly in malignant cell, by affecting the expression of key genes, such as oncogenes (e.g., c-myc and c-myb) and cyclins. In three human leukemic cell lines (HL-60, U937, and ML-1) [313

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