ROS homeostasis and metabolism

Tumor cells harbor genetic alterations that promote a continuous and elevated production of reactive oxygen species. Whereas such oxidative stress conditions would be harmful to normal cells, they facilitate tumor growth in multiple ways by causing DNA

damage and genomic instability, and ultimately, by reprogramming cancer cell metabolism. Both exogenous and endogenous sources of ROS production have been extensively described over the past decade(Panieri et al., 2015).The most biologically relevant are represented by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, professional enzymes that catalyze the production of O²⁻· or H2O2 using NADPH as a reductant (Lambeth 2004) and the mitochondrial electron transport chain (mETC), wherein mainly complexes I and II generate O2− through univalent reduction of molecular oxygen as a consequence of electron leakage during mitochondrial respiration using nicotinamide adenine dinucleotide (reduced form) (NADH) and FADH(Kussmaul et al., 2006; Quinlan et al., 2012). Detoxification from ROS is facilitated by non-enzymatic molecules (such as vitamin A, C, or E) or through antioxidant enzymes which specifically scavenge different kinds of ROS (Liou et al., 2010).

1.3.1 Nonenzymatic antioxidants

Nonenzymatic antioxidants include low-molecular-weight compounds, such as vitamins (vitamins C and E), β-carotene, uric acid, and GSH, a tripeptide (l-γ-glutamyl-l-cysteinyl-l-glycine) that comprise a thiol (sulfhydryl) group. Water-soluble vitamin C (ascorbic acid) provides intracellular and extracellular aqueous-phase antioxidant capacity primarily by scavenging oxygen free radicals. It converts vitamin E free radicals back to vitamin E and Lipid-soluble vitamin E is concentrated in the hydrophobic interior site of cell membrane and is the principal defense against oxidant-induced membrane injury (Mezzetti et al., 1996). β-Carotene inhibits the oxidant-induced NF-κB activation and interleukin (IL)-6 and tumor necrosis factor-α production. Carotenoids also affect

apoptosis of cells. Antiproliferative effects of RA have been shown in several studies (Donato et al., 2005; Niizuma et al., 2006). In addition, GSH is a cofactor for several detoxifying enzymes, such as GSH-Px and transferase. It has a role in converting vitamin C and E back to their active forms. GSH protects cells against apoptosis by interacting with proapoptotic and antiapoptotic signaling pathways (Masella et al., 2005).

1.3.2 Enzymatic antioxidants

The different type of enzymatic antioxidants is superoxide dismutase (SOD), enzymes catalase, and glutathione reductase, members of the aldehyde oxidoreductase and various peroxidases. These enzymes respectively dismutase superoxide radical, breakdown hydrogen peroxides and hydroperoxides to harmless molecules (H2O2/ alcohol and O2), thus they are important in the prevention of lipid peroxidation and maintaining the structure and function of cell membranes.

1.3.2.1 Superoxide dismutase

Superoxide dismutase (SOD) is the first detoxification enzyme and most powerful antioxidant in the cell. It is an important endogenous antioxidant enzyme that acts as a component of first line defense system against reactive oxygen species(ROS).

All 3 forms of SOD, that is, CuZn-SOD, Mn-SOD, and EC-SOD, are widely expressed in the human lung. Mn-SOD is localized in the mitochondria matrix. EC-SOD is primarily localized in the extracellular matrix, especially in areas containing high amounts of type I collagen fibers and around pulmonary and systemic vessels. It has also been detected in the bronchial epithelium, alveolar epithelium, and alveolar macrophages (Kinnula et al.,

2003; Kinnula 2005). Overall, CuZn-SOD and Mn-SOD are generally thought to act as bulk scavengers of superoxide radicals. The relatively high EC-SOD level in the lung with its specific binding to the extracellular matrix components may represent a fundamental component of lung matrix protection (Zelko et al., 2002). SOD enzyme deficiency is common. Hence, the enzyme is indispensable to cellular health, protecting body cells from excessive oxygen radicals, free radicals and other harmful agents that promote aging or cell death. The levels of SODs decline with age, whereas free radical formation increases. It has been suggested that proper daily SOD supplementation will protect the immune system and significantly reduce one’s chances of diseases and ultimately slow down aging process.

1.3.2.2 Catalase (CAT)

Catalase (CAT) is a 240 kilodalton (kDa) tetrameric protein with four similar subunits and is encoded by ctt1 gene mapping to chromosome 11(Radi et al., 1991).Each polypeptide subunit is 60 kDa in weight and contains a single ferriprotoporphyrin (Surai 2006).CAT is a common antioxidant enzyme present almost in all living tissues that utilize oxygen. Degradation of H2O2 is accomplished via the conversion between 2 conformations of catalase-ferricatalase (iron coordinated to water) and compound I (iron complexed with an oxygen atom), consequently completing the detoxification process imitated by SOD (Marklund 1984; Chelikani et al., 2004).It is abundant in cells, where it continuously scouts for hydrogen peroxide molecules. Catalase exists as a tetramer composed of 4 identical monomers, each of which contains a heme group at the active site. Each catalase can break down millions of hydrogen peroxide molecules in one second. CAT also reacts efficiently with hydrogen donors such as methanol, ethanol,

formic acid, or phenols with peroxidase activity. Hydrogen peroxide though at low amounts tends to regulate some physiological processes such as signaling in cell proliferation, cell death, carbohydrate metabolism, mitochondrial function, and platelet activation and maintenance of normal thiol redox-balance (Dröge 2002),however, at high concentrations it has been reported to be very deleterious to cells (Ercal et al., 2001).

Hence, the ability of CAT to effectively limit H2O2 concentration in cells underlines its importance in the aforementioned physiological processes as well as being a first line antioxidant defense enzyme. The deficiency or mutation of the enzyme has been linked with various disease conditions and abnormalities (Góth et al., 2004).

1.3.2.3 Glutathione reductase (GPx)

Glutathione Peroxidase (GPx) is an important intracellular enzyme that breakdown hydrogen peroxides (H2O2) to water; and lipid peroxides to their corresponding alcohols mainly in the mitochondria and sometimes in the cytosol (Góth et al., 2004). Most times, its activity depends on a micronutrient cofactor known as selenium. For this reason, GPX is often referred to as a selenocysteine peroxidase. The enzyme plays a more crucial role of inhibiting lipid peroxidation process, and therefore protects cells from oxidative stress (Gill et al., 2010). The clinical importance of GPx has been underlined by a number of studies. Chabory et al. (Chabory et al., 2009) postulated that individuals with lower GPx activity are predisposed to impaired antioxidant protection, which leads to oxidative damage to membrane fatty acids and functional proteins, and neurotoxic damage. Forgione and colleagues had previously hypothesized that GPX deficiency directly induces an increase in vascular oxidative stress, with attendant endothelial dysfunction (Forgione et al., 2002). Decreased GPx activity has also

been reported in tissues where oxidative stress occurs in several pathological animal models. The accumulation of increased levels of peroxide resulting from inactivation of GPx may act as a second messenger and regulate expression of antiapoptotic genes and the GPx itself to protect against cell damage. Glutathione peroxidases have also been implicated in the development and prevention of many common and complex diseases, including cancer and cardiovascular disease (Rayman 2005).

1.3.2.4 Aldehyde dehydrogenase

Aldehydes are generated during metabolism of various endobiotic and xenobiotic compounds. For example, aldehydes are associated with the metabolism of alcohols, amino acids (e.g., lysine, valine, proline and arginine), anticancer drugs (e.g., cyclophosphamide) and neurotransmitters (e.g., -aminobutyric acid (GABA), serotonin, noradrenaline, adrenaline, dopamine) (Vasiliou et al., 2004; Marchitti et al., 2007). Lipid peroxidation (LPO) of cellular phospholipids induces the formation of more than 200 highly-reactive aldehyde species, including 4-hydroxy 2-nonenal (4-HNE), malondialdehyde (MDA), 4-oxononenal (4-ONE), acrolein, crotonaldehyde and methylglyoxal (Ishitani et al., 1977; Marchitti et al., 2008; Zimniak 2011). Environmental pollutants, such as smog, cigarette smoke, motor vehicle exhaust, pesticides and various food additives, either contain or contribute to the formation of aldehydes, including formaldehyde, acetaldehyde and acrolein (Ishitani et al., 1977; Marchitti et al., 2008;

Zimniak 2011). While some aldehydes play vital roles in normal physiological processes, including vision, embryonic development and neurotransmission, many aldehydes are cytotoxic and carcinogenic (Yokoyama et al., 2001; Marchitti et al., 2008). ALDHs play critical roles in metabolizing these endogenous and exogenous molecules. Their

expression is up-regulated in response to abiotic and biotic stress generated by perturbed endobiotic and/or xenobiotic metabolism. Such stress-responsive expression of ALDHs manifests in a broad range of plant and animal species, underscoring the evolutionary conservation of biological adaptions to oxidative and electrophilic stresses. Aldehydes are strongly electrophilic, highly reactive and relatively long-lived compounds. Reactive aldehydes readily form adducts with DNA, RNA and proteins, leading to impaired cellular homeostasis, enzyme inactivation, DNA damage and cell death (Brooks et al., 2005; Jacobs et al., 2010). They have been implicated in oxidative stress-associated diseases, such as atherosclerosis, cancer, diabetes, chronic alcohol exposure, acute lung injury, and in neurodegenerative diseases like Alzheimer’s and Parkinson’s disease (Jacobs et al., 2010; Yin et al., 2011). The ALDH superfamily contains NAD(P)⁺ -dependent enzymes that oxidize a wide range of endogenous and exogenous aldehydes to their corresponding carboxylic acids (Vasiliou et al., 2004). The ability of ALDHs to act as ‘aldehyde scavengers’ is grounded in the observation that many have broad substrate specificities and can metabolize a wide range of chemically- and structurally-diverse aldehydes.

1.3.2.5 Peroxidases

Hydrogen peroxide and superoxide are also consumed by heme peroxidases, such as eosinophil peroxidase (EPO), lactoperoxidase (LPO), myeloperoxidase (MPO) (Valco M et al., 2007). Paradoxically, these enzymes are sources of more potent oxidants (hypochlorous acid, hydroxyl radical, singlet oxygen, etc.) that can cause a wide range of abnormalities including ischemic- related hypoxia and neuronal death (Everse et al., 2009). Significantly, MPO and EPO have been linked to various allergic, psychiatric, and

neurodegenerative diseases (Klebanoff 2005; Lefkowitz et al., 2008; Davis et al., 2008;

van der Veen et al., 2009).

1.3.2.6 Thioredoxin (Trx) reductase

Thioredoxin (Trx), originally identified as an electron donor for ribonucleotide reductase, functions to regulate various enzymes and trans-activating factors of genes, and is intimately involved in cell growth, differentiation, and death (Nakamura et al., 1997). Trx also functions as a protein disulfide isomerase that corrects disulfide bridges that are formed in error. Moreover, Trx directly donates electrons to peroxiredoxin and, hence, is directly linked to the peroxidase reaction (Fujii et al., 2002). After oxidation, an intramolecular disulfide bond is formed in Trx. Oxidized thioredoxin is reduced by thioredoxin reductase, a selenocystine-containing oxido-reductase, using NADPH as an electron donor. Since Trx-knockout mice are embryonically lethal (Matsui et al., 1996), Trx appears to play essential roles in the reproductive system and/or fetal development.

Among multiple roles of Trx, defect in electron donation to ribonucleotide reductase appears to be the main cause in Trx-knockout mice because DNA synthesis is essential for fetal development.

1.3.2.7 Aldo-keto reductase

Carbonyl compounds are produced by the oxidation of organic compounds, such as unsaturated fatty acids, and are highly reactive. They modify reactive sulfhydryl groups that are commonly present in redox-sensitive molecules, resulting in an impairment of the systems. Mammalians have several enzymatic systems that function to detoxify

carbonyl compounds. The aldo-keto reductase family includes enzymes that reduce carbonyl groups to alcohol using NADPH as an electron donor. Among the members of this family, aldose reductase, the AKR1B gene product, has been the most extensively studied because it is intimately involved in diabetic complications (Yabe-Nishimura 1998). An inhibitor of aldose reductase is one of proposed cures for diabetic complications. Aldehyde reductase, the AKR1A gene product, exhibits the highest similarity to aldose reductase among the family members (Jez et al., 1997) and appears to play a coordinate function (Iuchi et al., 2004). Since steroid hormones and their derivatives contain carbonyl groups and can serve as substrates for aldo-keto reductase (Wermuth et al., 1983), enzymes that are highly expressed in tissues with steroid hormone production may have a role in their elimination. The detoxification of carbonyls is activated by the binding of GSH, which indicates crosstalk between the GSH redox system and aldo-keto reductase system (Dixit et al., 2000).