The oxidation and subsequent β-cleavage of ARA by hydroxyl radicals (OH^•) yields 4-HNE, a nine carbon α, β-unsaturated aldehyde. 4-HNE is one of the major end products of lipid peroxidation and an important mediator of neural cell damage because of its ability to covalently modify biomolecules with disruption of important cellular functions (Esterbauer et al. 1991; Lin et al. 2005; Farooqui and Horrocks 2006) . The C3 position of 4-HNE is a highly reactive because of the presence of electeron withdrawing functional groups, the double bond. It serves as a site that undergoes a Michael addition reaction with cellular thiols cysteine, the imidizole nitrogen of histidine, and to a lesser extent, the amine nitrogen of lysine. The chemical reactions between 4-HNE and proteins include reactions between the C = C double bond with a nucleophile (Cys, glutathione (GSH) and amine) via 1,2- and 1,4-Michael addition. The 1,2-Michael addition involves the reaction of a primary amine (Lys) with the α,β-unsaturated carbonyl, resulting in the formation of a Schiff base at acidic pH (Petersen and Doorn 2004). 4-HNE not only inhibits DNA and RNA synthesis and disturbs calcium homeostasis, but also plays a substantial role in the disruption of the energy-producing capacity of mitochondria (Esterbauer et al. 1991; Guichardant et al. 2002). The localized concentration of 4-HNE may reach as high as 4.5 mM within the phospholipid bilayer. The half-life of 4-HNE is relatively short within cells, and 4-HNE rapidly removed by phase II reactions, allowing for excretion of water soluble conjugates (Gueraud et al. 2010) . Due to their amphiphilic nature, 4-HNE can easily diffuse across membranes and can covalently modify any protein in the cytoplasm and nucleus, far from their site of origin (Negre-Salvayre et al. 2008). Similarly, the 4-HNE formed outside the cells (i.e., in a site of inflammation or in plasma), can react with adjacent cells, even in cases when they are not primary sites of lipid peroxidation. 4-HNE modified proteins can also be removed by autophagic and proteasomal degradation pathways (Hill et al. 2008; Botzen and Grune 2007). The modification of adenine nucleotide translocator by 4-HNE causes the inhibition of enzymic activities and suppression of ADP and ATP transport through the inner mitochondrial membrane (Picklo et al. 1999). These events contribute to the disruption of the energy-producing capacity of mitochondria. In metabolic diseases, such as diabetes and metabolic syndrome, elevations in 4-HNE promote insulin resistance (Vincent et al. 2001; Russell et al. 2003). Moreover, at low doses, 4-HNE exerts an anti-cancer effect, by inhibiting cell proliferation, angiogenesis, cell adhesion and by inducing differentiation and/or apoptosis in various tumor cell lines (Pizzimenti et al. 2013). Collective evidence suggests that 4-HNE not only forms adducts with DNA (Esterbauer et al. 1991; Farooqui 2011; Chung et al. 2000), but also reacts with phospholipids (containing PUFAs such as linoleic and ARA) and nucleophilic amino acids (e.g cysteine, histidine and lysine residues (Farooqui 2011), making lipid membranes particularly vulnerable to 4-HNE modification .

The relative abundance of 4-HNE in the brain, vasculature, and adipose tissues depends not only on the rate of lipid peroxidation and 4-HNE synthesis, but also on the removal of 4-HNE adducts by phase II metabolic pathways such as glutathione-S-transferases. Thus, 4-HNE is metabolized by glutathione S-transferase A4 (GSTA4) producing glutathionyl-HNE (GS-HNE) and its metabolite glutathionyl-1,4-dihydroxynonene (GS-DHN). In 3T3-L1 adipocytes, high glucose levels and oxidative stress induce production of GS-HNE and GS-DHNE in a GSTA4-dependent manner and both glutathionylated metabolites induced secretion of TNFα from RAW264.7 and primary peritoneal macrophages. Targeted microarray analysis shows that GS-HNE and GS-DHN upregulates the expression of inflammatory genes (C3, C4b, c-Fos, igtb2, Nfkb1, and Nos2) supporting the view that GS-HNE and GS-DHN are pro-inflammatory glutathione metabolites of 4-HNE, which promote inflammation and insulin resistance in visceral tissues (Frohnert et al. 2013). Depending on its relative concentration, 4-HNE can induce a range of effects in the brain, vascular endothelial cells, adipose tissues, and smooth muscle cells. 4-HNE is also involved in carbonylation of adipocyte fatty acid-binding protein, a protein that has been implicated in the regulation of insulin resistance in vivo. In vitro, modification of adipocyte fatty acid-binding protein with 4-HNE has been mapped to Cys-117. Carbonylation of adipocyte fatty acid-binding protein results in10-fold reduction in fatty acid binding affinity for fatty acids. It is proposed that an increase in the carbonylation of a number of adipose-regulatory proteins may serve as a mechanistic link among increased oxidative stress, development of insulin resistance, and obesity (Grimsrud et al. 2007) .

Cross-linking of 4-HNE with glucose transporter, glutamate transporter, Na⁺, K⁺-ATPases, and NADP⁺-dependent isocitrate dehydrogenase produces changes in their activities (Mark et al. 1997; Keller and Mattson 1998; Lauderback et al. 2001; Yang et al. 2004). Inhibition of Na⁺, K⁺-ATPase by 4-HNE facilitates the opening of NMDA receptor channel and influx of calcium ions into the cell (Kadoya et al. 2003) (Fig. 7.3). This calcium entry can be very harmful for neurons. 4-HNE not only inhibits rat brain mitochondrial respiration, but also blocks neurite outgrowth, disrupts neuronal microtubules, and modifies cellular tubulin, which may contribute to the cytoskeletal changes in neurons undergoing a neurodegenerative process (Neely et al. 1999). In some cases, 4-HNE-mediated modification increases the activity of key regulatory proteins such as the dimerization and ligand-independent activation of the epidermal growth factor receptor (Liu et al. 1999) or the activation of the Nrf2 (nuclear factor erythroid 2-related factor 2) transcription factor, leading to increased expression of genes implicated in the antioxidant response (Zhang et al. 2006; West and Marnett 2005). In addition to altering the activity of enzymes, 4-HNE-mediated alkylation alters the rate of degradation of some proteins (alcohol dehydrogenase and αB-crystallin) (Carbone et al. 2004; Marques et al. 2004) (21, 22) .

As stated above, 4-HNE interacts with reduced glutathione to form GS-HNE complex, which is reduced to GS-1,4-dihydroxynonanol (GSDHN). This complex along with protein kinase signaling cascade facilitates the migration of NK-κB from cytosol to the nucleus, where it binds to the NFkB DNA binding sequence and transcribes various genes for proinflammatory cytokines , chemokines , proinflammatory enzymes, and other inflammatory markers (Fig. 7.4). The expression of a large number of genes involved in apoptosis, cell growth, survival, differentiation, and immune response is regulated by NF-κB, which is associated with an array of diseases such as neurotraumatic, neurodegenerative, and autoimmune diseases. The migration of NF-κB is the key for 4-HNE to modulate neuroinflammation . 4-HNE is also involved in neurogenic inflammation and pain through activation of irritant transient receptor potential ankyrin1 (TRPA1) channel (Trevisani et al. 2007). 4-HNE-mediated inactivation of thioredoxin and thioredoxin reductase through modification of cysteine and selenocysteine residues at the active site has also been linked to dysregulation of cellular redox status (Fang and Holmgren 2006).

Low levels of free 4-HNE (0.3 to 0.7 µM) have been reported to occur in the plasma of healthy individuals (Selley et al. 1989). 4-HNE levels increase significantly in plasma and tissues during aging (Gil et al. 2006) and in diseases associated with oxidative stress, such as atherosclerosis and diabetes, and neurological disorders (Farooqui 2011, 2013). Studies on the effect of 4-HNE on non-neural cells (vascular endothelial and smooth muscle cell) redox signaling and function have indicated that abundance of 4-HNE is found in the vasculature. Levels of 4-HNE in vasculature are not only dependent on the rate of lipid peroxidation and 4-HNE synthesis, but also on the removal of HNE adducts by phase II metabolic pathways such as glutathione-S-transferases (Farooqui 2011). Exposure of adipocytes to exogenous 4-HNE in a dose-dependent manner decreases in intracellular levels of adiponectin (Wang et al. 2012) . The expression of adiponectin gene expression is also elevated after 4-HNE treatment, with concomitant increase in peroxisome proliferator-activated receptor gamma (PPAR-γ) gene expression and transactivity. This effect can be abolished by T0070907, a PPAR-γ antagonist, supporting the view that PPAR-γ activation plays a critical role in this process (Wang et al. 2012). Based on studies on the effect of cycloheximide on adiponectin degradation, it is proposed that 4-HNE exposure enhances adiponectin protein degradation via ubiquitin-proteasome system. Collective evidence suggests that ARA-derived 4-HNE differentially regulates adiponectin gene expression and protein abundance and may play a mechanistic role in obesity-related plasma adiponectin decline (Wang et al. 2012). Exercise and dietary energy restrictions not only reduce 4-HNE levels, but also improve insulin sensitivity in muscle cells and increase levels of 4-HNE detoxifying systems (glutathione and oxidoreductases). Depending on its relative concentration, 4-HNE produces a range of hormetic effects in vascular endothelial and smooth muscle cells, including kinase activation, proliferation, induction of phase II enzymes and in high doses inactivation of enzymic processes and apoptosis (Farooqui 2011). 4-HNE also plays an important role in the pathogenesis of vascular diseases such as atherosclerosis, diabetes, and neurodegenerative diseases (Farooqui 2011) .

As stated above, IsoP are prostaglandin-like compounds generated by non-enzymic oxidation of esterified polyunsaturated fatty acids (arachidonic, eicosapentaenoic, adrenic acids) in neural membrane phospholipids. They are released by the action of cPLA₂ on peroxidized glycerophospholipids (Fig. 7.3) . The molecular mechanism by which isoprostanes are formed is analogous to the synthesis of prostaglandins by cyclooxygenases (Morrow et al. 1999). Unlike prostaglandins, which are formed from the COX, LOX, and EPOX-mediated oxidation of free ARA, the formation of isoprostanes in situ is initiated at the esterified ARA on the glycerophospholipid molecule (Fam and Morrow 2003). IsoPs are formed in situ in the phospholipid domain of cell membranes and circulating lipoproteins. They are then cleaved by phospholipases A₂, released extracellularly, circulate in blood and are excreted in urine.

Non-enzymic synthesis of the family of F₂-isoprostanes involves the formation of positional peroxyl radical isomers of ARA, which undergo 5-exo cyclization and a second molecule of oxygen adds to the backbone of the compound to form PGG₂-like compounds. These unstable bicycloendoperoxide intermediates are then reduced to the F₂-IsoPs. F₂-Isoprostane (F₂-IsoP) that is subsequently released in free form by the action of PLA₂ (Morrow et al. 1991; Fam and Morrow 2003; Montuschi et al. 2007) (Fig. 7.3). Another mechanism of isoprostane generation starts with a 4-exocyclization of a peroxyl radical leading to an intermediate dioxetane (Durand et al. 2005). Formation of F₂-IsoPs, E₂– and D₂-IsoPs can be obtained by rearrangement of H₂-IsoP endoperoxides. The occurrence of E₂/D₂-IsoPs has not been reported during ischemic injury (Farias et al. 2008). One of the E₂-IsoP isomers, 8-iso–PGE₂, which is produced abundantly in vivo, has a very potent biological activity and thus may contribute to brain damage associated with oxidative stress. As stated earlier, measurement of isoP is considered to be one of the most reliable index for assessing lipid peridation in vivo .

Although, IsoPs are initially formed in vivo esterified in phospholipids, but most studies exploring their bioactivity have been performed using unesterified IsoPs. 15-A_2t-IsoP is known to produce neurodegeneration in cultured neurons via mitochondrial ROS production, glutathione depletion, 12-lipoxygenase activation, and caspase cleavage (Musiek et al. 2006). Furthermore, 15-A_2t-IsoP also potentiates hypoxia-induced neuronal cell death, supporting the view that IsoP may be closely associated with the pathogenesis of ischemic injury (Zeiger et al. 2009). In brain microvasculature isoPs act as vasoconstrictors by inducing COX-mediated synthesis of thromboxane in endothelial cells (Lahaie et al. 1998). They induce their biochemical effects both via receptor-dependent and independent mechanisms. IsoP binds with TxA₂ receptors (TPRs) (Fig. 7.5). These receptors are not only found in the brain, but also in macrophages or monocytes, vascular endothelial cells, and platelets. TPRs modulate antiatherosclerotic, antivasoconstrictive, and antithrombotic effects, depending on the cellular target. TPR receptors mobilize intracellular Ca²⁺ and are coupled with the activation of protein kinases (MAP kinase and Ca²⁺/Rho kinase) (Kinsella et al. 1997; Pratico et al. 1997) . F₂-IsoP and its receptor also mediate their effects in vascular beds and platelet function by promoting interactions between endothelial cells and monocytes (Lahaie et al. 1998; Fam and Morrow 2003). Isoprostane-mediated monocyte adhesion does not dependent on VCAM-1, but involves protein kinases such as protein kinase A and mitogen-activated protein kinase kinase 1. F₂-IsoP also modulates the p38 MAPK pathway during monocyte adhesion (Cracowski 2004). Thus, F₂-IsoP not only affects vascular and bronchial smooth muscles function, but also modulates cellular proliferation (Fam and Morrow 2003). These processes may relate to inflammation and atherosclerosis. Receptor independent action of F₂-IsoP is due to adduct formation. These compounds contain reactive α,β-unsaturated carbonyl group on the prostane ring, which readily reacts with thiol-containing compounds to produce many biological effects. In isolated bovine retinae isoP produce dual effects. Low concentrations of 8-isoPGF_2α inhibit; whereas, higher concentrations of 8-isoPGF_2α stimulate K⁺-mediated [³H]D-aspartate overflow (Opere et al. 2005). Based on detailed investigations, it is suggested that both inhibitory and stimulatory effects are mediated via the activation of thromboxane receptors .

4-HNE-mediated protein carbonylation and treatment with cyclopentenone prostaglandin, 15-deoxy-Delta12,14-prostaglandin J₂ (15d-PGJ₂) are known to upregulate transcriptional activation of antioxidant-response genes . Thus, Nrf2, a central transcription factor involved in the regulation of antioxidant-responsive element-containing genes is activated in response to oxidative stress caused by the generation of 4-HNE and 15d-PGJ₂. The alkylation of the cytoplasmic inhibitor of Nrf2, Keap1 (Kelch-like ECH-associated protein 1), by 4-HNE and exposure of Nrf2-Keap1 complex to 15d-PGJ₂ mediate the dissociation of the Keap1-Nrf2 complex (Fig. 7.6). Once freed from inhibition by Keap1, Nrf2 migrates to the nucleus, where it interacts with antioxidant response elements (ARE) and facilitates the transcription of heme oxygenase-1 (HO-1), glutathione synthase (GS), NADP(H) quinine-oxidoreductase1 (NQO1), catalase and Na⁺-independent cystine/glutamate exchanger. This process results in strengthening of antioxidant defenses of neural cells (Fig. 7.6) (Levonen et al. 2004). In addition, 15d-PGJ₂ binds with peroxisome proliferator-activated receptor-gamma (PPAR-γ) and activates it in a dose-dependent manner by down-regulating NFκB (Ou et al. 2006). Both pathways decrease neuroinflammation (Giri et al. 2004). Post-infarct PGJ₂ administration produces neuroprotective effects following middle cerebral artery occlusion (MCAO) by non-overlapping NFκB and PPAR-γ dependent and independent mechanisms (Pereira et al. 2006; Lin et al. 2006) as well as being anti-inflammatory when used pre-induction in models of mixed stroke and in non-neuronal systems. Collective evidence suggests that 15d-PGJ₂ is effective in protecting against white matter ischemic in the brain .

Recently, another class of chemically reactive lipid peroxidation products has been identified: γ-ketoaldehyde isoketals (IsoKs) derived from ARA This γ-ketoaldehydes is much more reactive with cellular nucleophiles than 4-HNE and, unlike the structurally similar COX-derived levuglandins, IsoKs remain esterified to phospholipids. Thus, the synthesis of isoketals occurs through the rearrangement of H₂-IsoP endoperoxides. Isoketals differ from isoprostanes in containing a characteristic aldehyde group in a 1,4-dicarbonyl array, making them extremely reactive toward primary amino groups in proteins (Boutaud et al. 2005). Unlike F₂-IsoP, isoketals result in modification of biologically important proteins rather than activation of specific receptors (Davies et al. 2004). Isoketals are highly reactive γ-ketoaldehydes that form pyrrole adducts with the ε-amino group of lysine residues on protein (Davies et al. 2004). These pyrrole adducts are unstable in the presence of oxygen and are further transformed to lactam and hydroxylactam adducts, which accumulate as stable end products. IsoKs adduct almost instantaneously to protein lysine residues and readily induce protein-protein cross-linking. IsoK-mediated protein cross-linking induces impairment in proteasomal degradation of adducted proteins and inhibiting proteasome function. These processes may result in disruption of blood brain barrier and neuronal dysfunction, which may ultimately lead to dementia (Davies et al. 2004). In addition, isoketals have remarkable ability to crosslink proteins through oxidation of the pyrrole. Isoketals have been detected in tissues as well as biological fluids. In non-neural cells, IsoK also reacts with phosphatidylethanolamine (PTdEtn) and DNA (Sullivan et al. 2010; Carrier et al. 2009) . Exogenous addition of IsoK to human umbilical vein endothelial cells (HUVEC) produces approximately five-fold more abundant IsoK-PE adduct than protein adduct (Sullivan et al. 2010) Nothing is known about the levels and roles of IsoK-PE in vivo.