ROS perform important biological functions. Low levels of ROS are involved in signal transduction processes. H₂O₂, a potent oxidant freely diffuses across neural membranes to participate in intra- and intercellular signaling (Schrader and Fahimi 2006). Reversible protein oxidation by H₂O₂ is associated with signaling mechanism to modulate the activity of various kinases and phosphatases involved in the regulation of neural cell growth, proliferation, and apoptosis (Alder et al. 1999) Thus, serine/threonine kinases of the mitogen activated protein kinase (MAPK) family, including extracellular-regulated kinases (ERKs), c-Jun-NH₂-terminal kinase (JNK), and p38 MAPK are among the signaling molecules whose activity may be regulated by ROS-mediated post-translational modification (Alder et al. 1999). ROS also regulate phosphoinositol 3 kinase (PtdIns 3K)-Akt-p53 signaling pathway. Collective evidence suggests that low levels of ROS are needed for fundamental cellular functions, such as growth, adaptation responses, and for optimal functioning of the immune system. ROS-mediated signal transduction processes are supported by several transcription factors, such as nuclear factor kappaB (NF-κB) , activator protein-1 (AP-1) , and hypoxia-inducible factor (HIF)-1α (Valko et al. 2007) (Fig. 6.2). These transcription factors reside in cytoplasm. Production of high ROS in neural cells promotes the translocation of NF-κB from cytoplasm to the nucleus, where it interacts with NF-κB response element to facilitate the expression of proinflammatory enzymes (sPLA₂, COX-2, iNOS), cytokines (TNF-α, IL-1β, IL-6, IL-12) , chemokines (MIP-1α, MCPP1), growth factors, cell cycle regulatory molecules, adhesion molecule leading to inflammation (ICAM, VCAM, and E-selectin) and antiinflammatory molecules and adhesion molecules (Fig. 6.2). The DNA binding ability of NF-κB is modulated by redox status in the cell (Nishi et al. 2002). It is also shown that redox factor protein, Ref-1 reduces cysteine 62 in NFκB in the nucleus and this reaction is required for NF-κB binding to DNA (Nishi et al. 2002). Conversely, oxidation of this residue inhibits binding to DNA (Toledano and Leonardo 1991). In addition, glutathionylation of NFκB in the presence of ROS results in a decrease in its DNA binding ability and downstream transcriptional activity (Pineda-Molina et al. 2001).

Similarly, AP-1 is also involved in the increased expression of adhesion molecules and inflammatory cytokines. Transcription factor, HIF-1α not only activates a broad range of genes protecting cells against hypoxia, but also binds with p53 to regulate many genes including Bax (a proapoptotic member of the Bcl-2 family) (Gibson et al. 2001). Bax inactivates Bcl-2 by forming a heterodimer. The balance between levels of bcl-2 and bax can serve as an indicator of cell survival or death (Longoni et al. 1999).

Nuclear factor-erythroid 2-related factor 2 (Nrf2), a member of the cap“n”collar transcription factor family, is a master regulator of antioxidant defense genes and drug-metabolizing enzymes (Kensler et al. 2007). ROS are known not only to oxidize the cysteine residues of Keap1, but also upregulate Nrf2 through kinase activation. Subsequent phosphorylation of Nrf2 enhances Nrf2 dissociation from Kelch-like ECH-associated protein1 (Keap1) and allows migration of Nrf2 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 (Fig. 6.3) (Papaiahgari et al. 2006; Lee et al. 2003a; Lee et al. 2003b; Wang et al. 2012). Extra-cellular signal-regulated kinase (ERK), c-Jun N-terminal protein kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) pathways also regulate Nrf2 transcriptional activity. ERK and JNK regulate Nrf2 pathway activity positively, whereas p38 MAPK has been reported to regulate Nrf2 both positively and negatively (Naidu et al. 2009; Singh et al. 2010). ERK, JNK, and p38 MAPK act by phosphorylating N-terminal serine residues on Nrf2, which results as a response to electrophiles and oxidative stress (Huang et al. 2002). A 50 % reduction in cerebral blood flow following ischemic injury in mice results in activation of Nrf2 in neurons predominantly in cerebellar Purkinje cells and cingulate cortex (Liverman et al. 2004) supporting the view that activation of Nrf2 is closely associated with protection against neuronal injury. A variety of stimuli have been shown to induce Nrf2 activation by disrupting the Keap1-Nrf2 complex in the cytoplasm (Kaspar et al. 2009). Hydrogen peroxide and nitric oxide have been shown to oxidize several cysteine residues on Keap1, thereby forming intra- and intermolecular disulphides and inactivating Keap1 (Dhakshinamoorthy and Porter 2004). Nrf2 activation and its downstream signaling are important mediators of antioxidant signaling during exposure to low levels of ROS. Nrf2 also increases anti-inflammatory mediators, activity of the proteasome, and other transcription factors involved in mitochondrial biogenesis (Tufekci et al. 2011). Accumulating evidence suggests that activation of Nrf2 is associated with regulation of basal and inducible expression of numerous antioxidant stress genes, which play an important role in neuroprotection against oxidative stress not only in many animal models of neurotraumatic and neurodegenerative diseases, but also in human subjects.

Under pathological conditions or after exposure to certain toxic agents greater amounts of ROS are leaked from the mitochondrial electron transport chain, possibly at the sites of Complex I (NADH ubiquinone oxidoreductase) and Complex III (ubiquinone cytochrome c oxidoreductase) (Bailey et al. 1999). High levels of ROS not only suppress protein synthesis and activate proteases, but also attack phospholipids, cholesterol, protein, and DNA to generate uniquely oxidized biomolecules that can be used as “fingerprints” to detect oxidative stress in cells (Powers and Jackson 2008) (Fig. 6.4). High levels of ROS modulate telomeres (repeats of DNA–protein complexes), the structures, which are located at the ends of chromosomes and are essential for the stability of chromosome and cell replication. Telomeres are considered as a ‘biological clock’ of the cellular ageing. Telomerase is an enzyme that adds nucleotides to telomeric DNA thereby contributing to telomere maintenance, genomic stability, functions, and proliferative capacity of the cell. Increase in intracellular ROS levels is associated with acceleration in the rate of telomere shortening. The progressive shortening of telomeres leads to senescence, apoptotic cell death, or the oncogenic transformation of somatic cells in various tissues. Telomere length is modulated by various lifestyle factors (diet, exercise, calorie restriction, alcohol abuse, and smoking). It is known to determine overall health, lifespan, and the rate at which an individual ages (Epel et al. 2004; Minamino et al. 2008; Babizhayev et al. 2010; Lin et al. 2012). Recent in vitro studies have indicated that Mediterranean diet protects the cells from oxidative stress not only by preventing cellular senescence, and cellular apoptosis, but also through the maintenance of telomere length (Boccardi et al. 2013). In addition, mitochondria also become a main target of oxidative damage because of the relatively low levels of antioxidants, such as reduced glutathione (GSH), in mitochondria compared to cytosol (Fernandez-Checa et al. 1998).

ROS-mediated stimulation of sphingomyelinase (SMase) promotes the hydrolysis of sphingomyelin and generates ceramide, a lipid mediator that induces mitochondrial dysfunction, resulting in the production of more ROS and apoptotic cell death. Cholesterol oxides (oxysterols), which are derived from cholesterol by enzymic (cytochrome P450) and nonenzymic reactions, are markers of brain atrophy in patients with various neurodegenerative diseases (Leoni and Caccia 2011; Russell 2000; Schroepfer 2000). In brain, oxysterols have various neurochemical functions (induction of cell death, activation of inflammation, gene transcription, modulation of cognitive function, and/or oxidative processes). Therefore, oxysterols have been proposed to play major roles in the pathologies of various neurodegenerative diseases (Farooqui 2011). ROS, which are known to alter the intracellular redox balance, also facilitate the translocation of p53 to mitochondria. ROS along with 7-oxysterol and 7-ketocholesterol enhance mitochondrial translocation of Bax, mitochondrial membrane permeabilization, cytosolic release of cytochrome c, and cell death (Leoni and Caccia 2011; Russell 2000; Schroepfer 2000). In addition, 7-ketocholesterol also promotes the formation of ROS with subsequent activation of NFκB along with upregulation of interleukin-1β, interleukin (IL)-6, and IL-8 .

Direct measurement of ROS production is very difficult due to their high reactivity and low steady-state concentration. Nevertheless, the electron spin resonance (ESR) technique is a direct method of detection of species that have an unpaired electron, generally meaning that it is a free radical (Ashton et al. 1999). Common measures of bio-oxidation include the measurement of protein carbonyls as an indicator of protein oxidation; assessment of isoprostanes, malondialdehyde, and 4-hydroxyl-2-nonenol as signs of lipid peroxidation (Durand et al. 2005); and evaluation of DNA oxidation by assaying the levels of the oxidized base, 8-hydroxy-2′-deoxyguanosine (8-OH-dG) (see below) (Wink et al. 1991). The exogenous ROS burden creates an environment with oxidative stress mainly through the production of intracellular H₂O₂, which acts as a second messenger in cell signaling pathways. H₂O₂ reacts with freely accessible protein thiolate anions to form cyssulfenic acids, which is further oxidized to disulfide bonds. Most of these reactions are reversible in the presence of cellular thiol/disulfide systems such as GSH/GSSG, thioredoxin/thioredoxin reductase, and cysteine/cystine (Phillips et al. 2010). However, in the presence of excessive oxidation of cysteine and GSH depletion can lead to eventual overwhelming of endogenous oxidation-reduction (redox) reactions. Intracellular redox status is very important in protein function and to maintain cellular homeostasis. In neurotraumatic, neurodegenerative, and neuropsychiatric diseases enhanced ROS levels contribute to neuronal membrane damage not only by attacking neural cell membrane components (polyunsaturated fatty acids, sulfhydryl groups of proteins, bases of nucleic acids, and carbohydrates), but also by altering activities of various transcription factors (NF-κB, AP-1, HIF-1, and Nrf2) (Farooqui 2010). The effect of ROS-mediated damage to neural membrane phospholipids, sphingolipids, and cholesterol is cumulative and not amenable to repair, particularly in postmitotic cells such as neurons. Interactions between ROS and cellular components are known to alter cell membrane fluidity and permeability, ion transport, activities of membrane bound enzymes, protein cross-linking, inhibition of protein synthesis, and DNA damage eventually leading to apoptotic cell death (Farooqui and Horrock 2007). In addition, interactions between ROS and mitochondrial DNA (mtDNA) may lead to mutations, which may promote neuronal cell death. ROS also disrupt blood brain barrier (BBB) via matrix metalloproteinase (MMP) activation. The MMPs are zinc-containing enzymes, which are involved in the degradation of extracellular matrix around cerebral blood vessels and neurons (Gu et al. 2011). ROS-mediated activation of MMPs is involved in the stimulation of extracellular matrix degradation. This can occur at the tight junctions and therefore disrupt the vital BBB (Gu et al. 2012). It is also suggested that loss of BBB integrity due to ROS-mediated MMP activation is closely associated with cerebral ischemia – reperfusion injury (Gu et al. 2011; Gu et al. 2012).