Hydroxyl Radical (OH^∙)

Hydroxyl radical is the neutral form of hydroxide ion and is a highly reactive free radical [26]. It can strongly react with both organic and inorganic molecules including DNA, proteins, lipids, and carbohydrates and cause severe damage to the cells than any other ROS can do [27]. It is formed in a Fenton reaction (Eq. 2), in which H₂O₂ react with metal ions (Fe⁺² or Cu⁺), often bound in complex with different proteins such as ferritin (an intracellular protein that stores iron) and ceruloplasmin (plasma copper carrying protein) or other molecules [28]. Under stress conditions, an excess of O₂^•−releases free iron from ferritin and the released free iron participates in Fenton reaction to form OH^•. It is also formed by the reaction between superoxide radical and H₂O₂ in a reaction called Haber–Weiss reaction (Eq. 3) [29].

Peroxyl Radical (ROO^∙)

It is derived from oxygen in living systems. The simplest form of peroxyl radical is perhydroxyl radical (HOO^•) which is formed by the protonation of superoxide [30]. About 0.3 % of the total O₂^•− in the cytosol of a typical cell is in the protonated form. It initiates fatty acid peroxidation and also can promote tumor development [31].

Hydrogen Peroxide (H₂O₂)

Hydrogen peroxide is formed in vivo in a dismutation reaction catalyzed by the enzyme superoxide dismutase (SOD) (Eq. 1). It is not a free radical but it can cause damage to the cell at relatively low concentration (10 μM), but at higher levels, the cellular energy producing enzymes such as glyceraldehhyde-3-phosphate dehydrogenase are inactivated. It can easily penetrate the biological membranes. H₂O₂ has no direct effect on DNA but can damage DNA by producing hydroxyl radical (OH⁻) in the presence of transition metal ions [32]. The major antioxidant enzymes that can eliminate the H₂O₂ include catalase, glutathione peroxidase and peroxiredoxins [33, 34].

Singlet Oxygen (¹O₂)

It is an electronically high excited, meta-stable state of molecular oxygen and is a highly reactive toxic reactive oxygen species [35]. Upon activation, the molecular oxygen is excited to first state ¹Δ_g and then to next higher excited singlet state, 1εg. The first excited state, ¹Δ_g, has two electrons with opposite spins in the same π* orbital whereas, the second excited state, 1εg, has one electron in each degenerated π* orbital with opposite spins [36]. The ¹Δ_g state is extremely reactive, and compared to the other electronically excited states [36]. It is produced in vivo by the activation of neutrophils (Eq. 4) [37] and eosinophils [38]. It is also formed by some of the enzymatic reactions catalyzed by enzymes such as lipoxygenases [39], dioxygenases [40], and lactoperoxidase [41]. It is a highly potent oxidizing agent that can cause DNA damage [42] and tissue damage [38].

Ozone (O₃)

Ozone is a powerful oxidant may be produced in vivo by antibody catalyzed water oxidation pathway which plays an important role in inflammation [43]. It can form free radicals and other reactive intermediates by oxidizing the biological molecules. It can cause lipid peroxidation [44] and oxidize different functional groups, for example, amine, alcohol, aldehyde and sulphydryl, present in proteins [45, 46] and nucleic acids [47]. It can also cause chromosomal aberrations which may be due to direct attack by O₃ or by the free radicals generated by it [48].

Hypochlorous Acid (HOCl)

It is a major oxidant produced by the activated neutrophils at the site of inflammation from hydrogen peroxide and chloride in a reaction catalyzed by the enzyme myeloperoxidase [49].

HOCl is a strong reactive species involved in oxidation and chlorination reactions. It can oxidize thiols and other biological molecules including, ascorbate, urate, pyridine nucleotides, and tryptophan [50, 51]. HOCl chlorinates several compounds such as amines to give chloramines; tyrosyl residues to give ring chlorinated products, cholesterol and unsaturated lipids to give chlorohydrins, and it can also chlorinate DNA [52].

Nitric Oxide or Nitrogen Monoxide (NO^•)

It is a small molecule generated in tissues by different nitric oxide synthases (NOS)s which convert L-arginine to L-citrulline [53]. In this reaction one of the terminal guanido nitrogen atoms undergo oxidation and produces NO^.. Three types of isoforms of NOS such as neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) are involved in the formation of the NO radical.

It is both aqueous and lipid soluble and therefore it readily diffuses through cytoplasm and plasma membrane [54]. The NO^• is an important intracellular second messenger stimulates guanylate cyclase and protein kinases and helps in smooth muscle relaxation in blood vessels. It is identical to endothelium derived relaxing factor (EDRF) produced by vascular endothelial cells which is an important mediator of vascular responses [55]. It can also act as an important cellular redox regulator [56] and regulate enzymatic activity by nitrosylating the proteins [57]. Since it is involved in many biological activities like blood pressure regulation, smooth muscle relaxation, neurotransmission, defensive mechanisms and immune regulation, this molecule was regarded as molecule of the year 1992 [58].

Mitochondria

Most of the intracellular ROS are derived from mitochondria (Fig. 1). The superoxide radicals are produced at two major sites in the electron transport chain, namely complex I (NADH dehydrogenase) and complex III (ubiquinone cytochrome c reductase). The transfer of electrons from complex I or II to coenzyme Q or ubiquinone (Q) results in the formation of reduced form of coenzyme Q (QH₂). The reduced form QH₂ regenerates coenzyme Q via an unstable intermediate semiquinone anion (^∙Q^-) in the Q-cycle. The formed ^∙Q^- immediately transfers electrons to molecular oxygen leading to the formation of superoxide radical. The generation of superoxide is non-enzymatic and therefore higher the metabolic rate, the greater is the production of the ROS [63].

The superoxide anion is converted to hydrogen peroxide by the action of mitochondrial superoxide dismutase (MnSOD). H2O2 can be detoxified by the Catalase (CAT) and glutathione peroxidase (GPx).

The other mitochondrial components which contribute to the formation of ROS include monoamino oxidase, α-ketoglutarae dehydrogenase, glycerol phosphate dehydrogenase and p66shc [64].

The p66Shc is an important member of the ShcA protein family, which contains another two more proteins, p46Shc and p52 Shc. The mammalian p66Shc is a 66-kDa isoform of the growth factor adaptor protein involved in apoptosis. It mediates the production of ROS in mitochondria. Most of the p66Shc is located in cytoplasm with a small fraction localized in the mitochondrial intermembrane space. Upon oxidative stress, p66Shc translocates to mitochondrial intermembrane space, where it associates with cytochrome-c, thus inducing ROS generation [65].

Peroxisomes

In peroxisomes the respiratory pathway involves the transfer of electrons from various metabolites to the oxygen leads to H₂O₂ formation [66], but is not coupled to oxidative phosphorylation to produce ATP instead free energy is released in the form of heat. The other free radicals produced in peroxisomes include H₂O₂, O₂^•− OH^• and NO^•. The β-oxidation of fatty acids is the major metabolic process producing H₂O₂ in the peroxisomes. As reviewed elsewhere, the different peroxisomal enzymes such as acyl CoA oxidases, D-amino acid oxidase, L-α-hydroxy oxidase, urate oxidase, xanthine oxidase, D-aspartate oxidase have been shown to produce different ROS [67] [see Table 2].

Endoplasmic Reticulum

The enzymes of endoplasmic reticulum such as cytochrome p-450 and b5 enzymes and diamine oxidase contribute to the formation of ROS [68]. Another important thiol oxidase enzyme, Erop1p catalyses the transfer of electrons from dithiols to molecular oxygen results in the formation of H₂O₂ [69].

The other endogenous sources of ROS include prostaglandin synthesis, auto-oxidation of adrenalin, phagocytic cells, reduced riboflavin, FMNH₂, FADH₂, cytochrome P _450, immune cell activation, inflammation, mental stress, excessive exercise, infection, cancer, aging, ischemia etc. [68].

On the other hand, ROS are also produced in the biological systems by various exogenous sources shown in Table 3 [10].

Molecular targets of free radicals

When there is an imbalance between the free radical production (ROS/RNS) and antioxidant defenses, the former will be produced in higher concentrations leading to oxidative stress and nitrosative stress. Since these free radicals are highly reactive, they can damage all the three important classes of biological molecules including nucleic acids, proteins, and lipids [70].

Deoxyribonucleic Acid (DNA)

Both ROS/RNS can oxidatively damage the nucleic acids. The mitochondrial DNA is more vulnerable to the ROS attack than the nuclear DNA, because it is located in close proximity to the ROS generated place. ROS, most importantly, the OH^• radical directly reacts with all components of DNA such as purine and pyrimidine bases, deoxyribose sugar backbone [71] and causes a number of alternations including single and double stranded breaks in DNA. The OH^∙ radical abstracts hydrogen atoms to produce a number of modified puine as well as pyrimidine base by-products and DNA- protein cross links. The pyrimidine attack by OH^• produces different pyrimidine adducts like thymine glycol, uracil glycol, 5-hydroxydeoxy uridine, 5-hydroxy deoxycytidine, hydantoin and others. The purine adducts formed by hydroxyl radical attack include, 8-hydroxydeoxy guanosine, 8-hydroxy deoxy adenosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine. The other free radical induced adducts of DNA bases include, 5-formyl uracil, cytosine glycol, 5,6-dihydrothyronine, 5-hydroxy-6-hydro-cytosine, 5-hydroxy-6-hydro uracil, uracil glycol, and alloxan [72]. The major free radical induced adducts of the sugar moiety in DNA include glycolic acid, 2-deoxytetrodialdose, erythrose, 2-deoxypentonic acid lactone, 2-deoxypentose-4-ulose [72]. 8-hydroxy deoxyguanosine is considered as the biomarker of oxidative DNA damage and is involved in mutagenesis, carcinogenesis and ageing. The levels of 8-OHdG are higher in mitochondrial DNA than in nuclear DNA [73].

On the other hand, the RNS, most importantly, peroxynitrite (OONO⁻) interacts with guanine to produce nitrative and oxidative DNA lesions such as 8-nitroguanine and 8-oxodeoxyguanosine respectively [74]. 8-nitroguanine formed is unstable and can be spontaneously removed, resulting in the formation of an apurininc site [75]. Conversely adenine can be paired with 8-nitroguanine during DNA synthesis resulting in a G-T transversions [76]. Accordingly 8-nitroguanine is a mutagenic DNA lesion involved in carcinogenesis.

Ribonucleic acid (RNA)

ROS can attack different RNAs produced in the body. The RNA is more prone to oxidative damage than DNA, due to its single stranded nature, lack of an active repair mechanism for oxidized RNA, less protection by proteins than DNA and moreover these cytoplasmic RNAs are located in close proximity to the mitochondria where loads of ROS are produced. Indeed, RNA is subjected to more oxidative damage than DNA in humans [77]. 7, 8-dihydro-8-oxo-guanosine (8-oxoG) is the most extensively studied RNA damage product and its levels are raised in various pathological conditions like Alzheimer’s disease [78], Parkinson’s disease [79], atherosclerosis [80], hemochromastosis [81] and myopathies [82].

Lipids

The membrane lipids, especially the polyunsaturated fatty acid residues of phospholipids are more susceptible to oxidation by free radicals [83]. The lipid peroxidation is very important in vivo because of its involvement in various pathological conditions. The lipid peroxidation results in the loss of membrane functioning, for example, decreased fluidity, inactivation of membrane bound enzymes and receptors [84]. The lipid peroxidation is initiated, when any free radical attacks and abstracts hydrogen from a methylene groups (CH₂) in a fatty acid (LH) which results in the formation of a carbon centered lipid radical (L^•). The lipid radical can react with molecular oxygen to form a lipid peroxyl radical (LOO^•). The resultant lipid peroxyl radical (LOO^•) undergo rearrangement via a cyclisation reaction to form endoperoxides, which finally form malondialdehyde (MDA) and 4-hydroxyl nonenal (4-HNA), the toxic end products of lipid peroxidation that cause damage to the DNA and proteins [85]. These lipid peroxyl radicals can further propagate the peroxidation process by abstracting hydrogen atoms from the other lipid molecules. Isoprostanes (prostaglandin like substances produced by in the body by the esterfication of arachidonic acid) constitute the important product of lipid peroxidation of arachidonic acid and are considered as the makers of the oxidative lipid damage [86].

Neurodegenrative Diseases

The central nervous system (CNS) is particularly susceptible to the oxidants due to the presence of high lipid content, high consumption of oxygen, and low levels of antioxidant enzymes, for example, SOD is localized primarily in neurons, and GSH and GPx are localized in astrocytes [109]. The lipid peroxidation by ROS leads to progressive loss of membrane fluidity, decreases membrane potential, and increases permeability to ions such as Ca²⁺. The regions of the brain such as hippocampus, substantia nigra, and the striatum are particularly susceptible to attack by free radicals [110, 111]. The oxidative-stress state has been also implicated in several neurodegenerative diseases such as Alzheimer’s [112], Parkinson’s [113], Huntington’s, lateral amyotrophic sclerosis [114], and multiple Sclerosis [115].

Parkinson’s Disease (PD)

Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons (involve in learning, memory and motor control), especially in the midbrain area called the substantia nigra, accompanied by deposition of inclusion bodies (Lewy bodies) of α-synuclein. The redox imbalance causes oxidative damage to these neurons and begins to alter the synthesis and metabolic pathway of dopamine leads to a further increase in oxidative stress because of quinine formation [111]. The characteristic clinical symptoms of PD include, jerky movements, trembling of the hands and lips, and tremors [116]. Dopamine, a neurotransmitter, can also act as a metal chelator, has the ability to generate H₂O₂ via Fenton reaction. Ceruloplasmin (an extracellular ferroxidase required for regulating cellular iron load and transport) oxidation results in the decreased ferroxidase activity followed by the accumulation of intracellular iron in neurons in PD. The increased levels of Fe⁺³ mediate the production of hydroxyl radicals, results in the damage of dopaminergic neurons in PD [117].

Alzheimer’s Disease (AD)

Alzheimer’s disease (AD) is characterized by the accumulation of amyloid protein plaques (formed from the improper folding and processing of amyloid β precursor protein-ABPP) [168] and intracellular neurofibrillary tangles made up of abnormal and hyperphosphorylated tau protein [118]. The hyperphosphorylated tau protein aggregates binds to Fe³⁺, results in the production of neurofibrillary tangles [119]. The Amyloid-β peptide (Aβ) can chelate with transition metal ions (Cu²⁺, Zn²⁺ and Fe³⁺), and produce H₂O₂ via transition metal mediated catlysis and finally gives toxic OH˙ radical [120]. The lipid peroxidation is also extensive in AD patients, which can induce neuronal death by multiple mechanisms such as impairment of function of ion pumps (both Na⁺/K⁺-ATPase and Ca⁺²-ATPase), glucose transporters and glutamate transporters. The other oxidative markers of protein damage such as protein carbonyls and 3-nitrotyrosine have been also observed in AD patients [118].

Colorectal Cancer

Colorectal cancer (CRC) is the third most common cancer worldwide, accounting for 608,000 deaths per year [129]. The gastrointestinal tract, particularly the colon and rectum, is continuously exposed to ROS originating from both endogenous and exogenous sources [130]. Colon cancer originates from the epithelial cells that line the bowel. These cells divide rapidly and have a high metabolic rate [131]. Since the intestinal mucosa is constantly confronting with diet and bacterial-derived oxidants and carcinogens, an unrestrained production of free radicals, redox imbalance, and DNA damage occurs, finally leads to an altered intestinal metabolic homeostasis with cancer as an endpoint [133]. The human colorectal tumors have increased levels of nitric oxide (NO) [132], 8-oxodG in DNA [133], and lipid peroxides [134]. Suzuki et al. 2004 [135] have reported increased serum levels of oxidized low density lipoprotein in patients with CRC compared to healthy individuals.

Breast Cancer

Damage to the breast epithelium by ROS can lead to fibroblasts proliferation, hyperplasia of epithelium, cellular atypia and breast cancer [136]. In majority of breast carcinomas the oxidative stress can be induced by the over expression of thymidine phosphorylase enzyme which catabolizes thymidine to thymine and 2-deoxy-D-ribose-1-phosphate; the latter is a powerful reducing sugar that rapidly glycates proteins, generating oxygen radicals within the carcinoma cell [137]. Another breast specific mechanism of oxidative stress induction involves a mammary gland specific lactoperoxidase enzyme catalyzed one electron oxidation of 17-β-oestradiol to a reactive phenoxyl radical [138].

Prostate Cancer

The ROS produced are responsible for the cellular proliferation of prostate cancer cells [139]. Overexpression of NADPH oxidase 1 (Nox1) protein is an early event in the development of prostate cancer. Prostrate Tumors have considerably higher levels of ROS and Nox1 levels [140]. The superoxide produced (by NOX) in prostate cancer cells facilitates cellular immortality through resistance to programmed cell death which results in cancer-promoting effect [141]. Kumar et al. 2008 [142] have reported elevated ROS levels in prostate cancer cells compared with normal cells. Veeramani et al. 2008 [143] have reported that elevated levels of ROS might also be formed by the simultaneous increased levels of p66Shc protein in prostate cancer cells.

Lung Cancer

Lung cancer has been the most commonly diagnosed cancer and is the central cause of cancer death in men worldwide [144]. Lung cancer mortality account 30 % of all cancer related deaths. Oxidative stress plays an important role in lung inflammation and lung cancer [145]. Cigarette Smoking is the most crucial environmental risk factor in lung cancer etiology. It is estimated that smoking accounts for ~80 % of global lung cancer burden in males and 50 % in females [146]. Cigarette smoke particulate matter contains complex mixture of numerous carcinogens and stable ROS with very long half-lives. These ROS can damage the tissues resulting in progressive transformation of cells into the malignant form, which leads to increased frequency of mutations by the oxidative damage to DNA and, eventually leading to lung cancer [147]. Smokers develop lung cancer a 10-fold higher than non-smokers. Lung cancer (LC) and chronic obstructive pulmonary disease (COPD) commonly coexist in smokers, and the presence of COPD increases the risk of developing LC [148].

Bladder Cancer

Bladder cancer is one of the most common cancers across the world, ranking the fourth and tenth in men and women, respectively [149]. The most common risk factors for bladder cancer are cigarette smoking, exposure to industrial carcinogens (aromatic amines), high levels of arsenic intake and diet [150]. Oxidative stress critically contributes to the development of bladder cancer [151]. Various lines of evidence reported an increased oxidative stress in patients with breast cancer [152, 153]. Increased NO levels have been reported in bladder cancer patients [154]. This NO stimulates matrix metalloproteinases (MMPs), especially prolidase activity, which is involved in the terminal step of collagen degradation. Significantly higher serum prolidase activities were reported in patients with bladder cancer than healthy controls [155]. Therefore, increased prolidase activity may, in part, play a role in the pathogenesis of bladder cancer.

Epidemiological studies reveal that low levels of antioxidants are associated with an increased risk of cancer. Significant increase in total oxidant status levels and decrease in total antioxidant status were observed in patients with bladder cancer [156]. Significantly lower levels of plasma protein, total thiol groups and protein-bound thiol groups and elevated levels of Protein carbonyl groups were observed in bladder cancer patients than in healthy controls [157].

Cardiovascular Diseases (CVDs)

Cardiovascular diseases are a class of pathologies involving the heart and blood vessels (arteries, capillaries, and veins). They include cardiac diseases, vascular diseases of the brain and kidney, and peripheral arterial disease. Most of the people are dying due to CVDs compared to other diseases [158].

Atherosclerosis

Atherosclerosis is a condition commonly referred to as hardening of the arteries. Hyperlipidemia is a major risk factor for atherosclerosis. Elevated levels of oxidized low density lipoprotein (LDL), glucose and free fatty acids are found in patients with atherosclerosis, T2D, and obesity [159]. A profound imbalance of oxidants and antioxidants resulting in oxidative stress is observed in atherosclerosis. In the vessel wall, endothelial cells, smooth muscle cells (SMCs) and macrophages are sources of free radicals [160]. Endothelial dysfunction leads to increased endothelial permeability, up regulation of endothelial adhesion molecules, and inflammatory cell infiltration into the arterial wall [160]. A substantial data has been shown that ROS are involved in endothelial injury, dysfunction, and lesion progression [161]. The ROS dependent activation of the MMPs results in the degradation of intimal extracellular matrices and promotes smooth muscle cell migration [162]. Cigarette smoking contain large amount of free radicals and may down-regulate key exogenous and endogenous antioixdants such as vitamin-D, carotenes, GPx and SOD and can lead to the dysfunction of monocytes and vascular smooth muscle cells [163]. The proatherogenic agents such as oxidaised lipids, high glucose and cigarette constituents give rise to increased free radical production.

The endothelial nitric oxide synthase (eNOS) derived NO^. plays an important role in maintaining vascular tone and vasoreactivity, vasodialation, platelet aggregation and in maintaining balance between smooth muscle cell growth and differentiation. Decreased NO bioavailabilty is the one of the major feature of the CVDs [249]. During reduced availability of BH4 or L-Arg, eNOS becomes uncoupled from a NO^∙ to a O2∙- state [164]. The O₂^•− formed can interact with NO^• to produce ONOO^-, a damaging and cytotoxic free radical with potential to disturb cardiovascular function. ONOO^- reduces the bioavailability of NO leading to reduced endothelial vascular regulatory capacity and increased vascular dysfunction. ONOO⁻ also inactivates the BH4 cofactor effectively amplifying the damaging effects of eNOS uncoupling the endothelium [164]. Mitochondrial DNA damage is frequently observed in human atherosclerosis in both circulating and vessel wall cells [165]. Oxidative stress mediated damaged mitochondrial DNA that escape autophagy induces a potent inflammatory response in atherosclerosis [166]. Malfunction of DNA repair leads to defects in cell proliferation, apoptosis, and mitochondrial dysfunction, which in turn leads to ketosis, hyperlipidemia, and increased fat storage, promoting atherosclerosis and the metabolic syndrome [167].