**2. Formation of reactive oxygen species**

**Oxygen (**O2**)** is an element obligatory for life and living systems have evolved to survive in the presence of molecular O2. Oxidative properties of O2 play a vital role in diverse biological phenomena. O2 has double-edged properties, being essential for life; it can also aggravate the damage within the cell by oxidative events. This situation is referred to as the "Oxygen Paradox" [Sen et al., 2010; Khanna & Shiloh, 2009].

Aerobic organisms are constantly subjected to a variety of reactive entities derived from molecular O2, often collectively referred to as reactive oxygen species (ROS). Some ROS contain unpaired electrons and are therefore referred to as free radicals. A radical is an atom or group of atoms that have one or more unpaired electrons. Radicals can have positive, negative or neutral charge. A prominent feature of radicals is that they have extremely high chemical reactivity, which explains not only their normal biological activities, but also how they inflict damage on cells. There are many types of radicals, but in biological systems most significant are those derived from O2 [Khanna & Shiloh, 2009, Colton & Gilbert, 1999]. The radicals derived from the reduction of molecular oxygen: superoxide/hydroperoxyl radicals hydrogen peroxide and the hydroxyl radical (•OH) the species derived from the reaction of carbon-centered radicals with molecular oxygen: peroxyl radicals alkoxyl radicals and organic hydroperoxides (ROOH); and other oxidants resulting in free radical formation such as hypochlorous acid (HOC1), peroxynitrite and singlet O2 [Khanna & Shiloh, 2009, Colton & Gilbert, 1999].

The **diatomic oxygen** molecule qualifies as a radical, because it possesses two unpaired electrons, each located at a different orbital. Since both electrons have the same quantum spin number, O2 itself has relatively low reactivity. Another radical derived from O2 is singlet oxygen, 1O2. This is an excited form of O2 in which one of the electrons jumps to a superior orbital following absorption of energy. For O2 to oxidize a molecule directly, it would have to accept a pair of electrons with a spin opposite to that of the O2. In biological systems, O2 can accept an electron and form one of the following species: superoxide anion (•O2−), •OH, or hydrogen peroxide (H2O2). These molecules possess various degrees of reactivity with nonradical compounds [Colton & Gilbert, 1999, Kohen & Nyska, 2002].

The mitochondrial respiratory chain is the major source of •O2−, [Colton & Gilbert, 1999, Kohen & Nyska, 2002, Bielski & Cabelli, 1995, Halliwell & Gutteridge, 1999, Schafer & Buettner, 2001, Forman & Boveris, 1982]. •O2− is abundant and can reach an intracellular concentration of about 10−11 M [Halliwell & Gutteridge, 1999, Schafer & Buettner, 2001, Forman & Boveris, 1982, Babior, 2000, Nohl & Hegner, 1978]. •O2− is not highly reactive for biological molecules however once formed it quickly undergoes dismutation to generate hydrogen peroxide, which is highly reactive. This reaction is markedly accelerated by a family of enzymes, the superoxide dismutases (SODs). •O2− can react with H+ to form HO2• (hydroperoxy radical) which is much more reactive than •O2−. NADPH oxidase, primarily located in phagocytes, neutrophils and monocytes, can generate •O2− and other reactive oxidants that are used for fighting invading microorganisms [Sohal, 1997].

If the mechanisms of oxidative DNA damage and DNA repairing system are well understood, the diseases resulting from oxidative DNA damage or inefficient DNA repairing could be treated in future and a better understanding of these mechanisms would

**Oxygen (**O2**)** is an element obligatory for life and living systems have evolved to survive in the presence of molecular O2. Oxidative properties of O2 play a vital role in diverse biological phenomena. O2 has double-edged properties, being essential for life; it can also aggravate the damage within the cell by oxidative events. This situation is referred to as the

Aerobic organisms are constantly subjected to a variety of reactive entities derived from molecular O2, often collectively referred to as reactive oxygen species (ROS). Some ROS contain unpaired electrons and are therefore referred to as free radicals. A radical is an atom or group of atoms that have one or more unpaired electrons. Radicals can have positive, negative or neutral charge. A prominent feature of radicals is that they have extremely high chemical reactivity, which explains not only their normal biological activities, but also how they inflict damage on cells. There are many types of radicals, but in biological systems most significant are those derived from O2 [Khanna & Shiloh, 2009, Colton & Gilbert, 1999]. The radicals derived from the reduction of molecular oxygen: superoxide/hydroperoxyl radicals hydrogen peroxide and the hydroxyl radical (•OH) the species derived from the reaction of carbon-centered radicals with molecular oxygen: peroxyl radicals alkoxyl radicals and organic hydroperoxides (ROOH); and other oxidants resulting in free radical formation such as hypochlorous acid (HOC1), peroxynitrite and singlet O2 [Khanna & Shiloh, 2009, Colton

The **diatomic oxygen** molecule qualifies as a radical, because it possesses two unpaired electrons, each located at a different orbital. Since both electrons have the same quantum spin number, O2 itself has relatively low reactivity. Another radical derived from O2 is singlet oxygen, 1O2. This is an excited form of O2 in which one of the electrons jumps to a superior orbital following absorption of energy. For O2 to oxidize a molecule directly, it would have to accept a pair of electrons with a spin opposite to that of the O2. In biological systems, O2 can accept an electron and form one of the following species: superoxide anion (•O2−), •OH, or hydrogen peroxide (H2O2). These molecules possess various degrees of reactivity with nonradical compounds [Colton & Gilbert, 1999, Kohen

The mitochondrial respiratory chain is the major source of •O2−, [Colton & Gilbert, 1999, Kohen & Nyska, 2002, Bielski & Cabelli, 1995, Halliwell & Gutteridge, 1999, Schafer & Buettner, 2001, Forman & Boveris, 1982]. •O2− is abundant and can reach an intracellular concentration of about 10−11 M [Halliwell & Gutteridge, 1999, Schafer & Buettner, 2001, Forman & Boveris, 1982, Babior, 2000, Nohl & Hegner, 1978]. •O2− is not highly reactive for biological molecules however once formed it quickly undergoes dismutation to generate hydrogen peroxide, which is highly reactive. This reaction is markedly accelerated by a family of enzymes, the superoxide dismutases (SODs). •O2− can react with H+ to form HO2• (hydroperoxy radical) which is much more reactive than •O2−. NADPH oxidase, primarily located in phagocytes, neutrophils and monocytes, can generate •O2− and other reactive oxidants that are used for fighting invading

also allow biomarkers of DNA damage to become potentially useful clinical tools.

**2. Formation of reactive oxygen species** 

& Gilbert, 1999].

& Nyska, 2002].

microorganisms [Sohal, 1997].

"Oxygen Paradox" [Sen et al., 2010; Khanna & Shiloh, 2009].

The **•OH** is an extremely reactive oxidant [Halliwell & Gutteridge, 1999, Khanna & Shiloh, 2009]. It is also a short-lived molecule with an estimated half-life of nanoseconds at 37◦C, traveling only a few Ångstroms. Despite its short life span, •OH is capable of inducing considerable damage to nuclear and mitochondrial DNA. This radical alone can cause over 100 types DNA modifications [Khanna & Shiloh, 2009, Michalik et al., 1995]. In addition, •OH can lead to lipid peroxidation and oxidation of amino acids, sugars, and metals. The •OH is a major product of irradiation due to radiation-induced dissociation of water molecules.

Although **H2O2** itself is not a radical, it is included in ROS due to producing highly reactive free radical, •OH. H2O2 is one of the most stable ROS and acts as a messenger in cellular signaling pathways [Khanna & Shiloh, 2009, Kamata & Hirata, 1999]. There are some enzymes that can produce H2O2 directly or indirectly, including SOD, monoamine oxidase (MAO), diamine and polyamine oxidase, and glycolate oxidase. Under normal conditions, H2O2 is not toxic up to a cellular concentration of about 10−8 M [Imlay et al., 1988] H2O2 molecules are freely dissolved in aqueous solution and can easily penetrate biological membranes. Their deleterious chemical effects can be divided into the categories of direct activity, originating from their oxidizing properties, and indirect activity in which they serve as a source for more deleterious species, such as OH. or HClO. In the presence of transition metals such as Fe2+ or Cu+, H2O2 it can be converted to highly reactive •OH, either by Fenton or Harber– Weiss reactions [Yamasaki & Piette, 1991, Halliwell & Gutteridge, 1999, Khanna & Shiloh, 2009]. H2O2 is detoxified by a set of enzymes that includes the selenium-dependent glutathione peroxidase (GPx) and catalase.

**The nitric oxide (NO), or nitrogen monoxide**, which is a radical (NO• ), is produced by the oxidation of one of the terminal guanido nitrogen atoms of L-arginine. In this reaction, Larginine is converted to NO and L-citrulline by nitric oxide synthase (NOS) which has three isoforms: neuronal NOS, endothelial NOS (eNOS), and inducible NOS (iNOS). NO is quite stable and benign for a free radical, with a lifetime of several seconds. Under normal conditions, NO has many physiological functions such as a neuronal messenger and modulator of smooth muscle contraction. NO can interact with •O2− to form the peroxynitrite anion (ONOO−) that induces a cascade of events that can eventually lead to cell death [Radi et al., 1991a]. This molecule accounts for much of the NO toxicity. The reactivity of ONOO− is roughly the same as that of •OH and N O2•. Its toxicity is derived from its ability to directly nitrate and hydroxylate the aromatic rings of amino acid residues [Schafer & Buettner, 2001] and to react with sulfahydryls [Beckman et al., 1992], lipids [Radi, 1991b], proteins [Moreno & Pryor, 1992] and DNA [King et al., 1992]. Under physiological conditions, ONOOH can react with other components present in high concentrations, such as H2O2 or CO2, to form an adduct that might be responsible for many of the deleterious effects seen in biological sites. Peroxynitrite anion can also affect cellular energy status by inactivating key mitochondrial enzymes [Radi et. Al, 1994], and it may trigger calcium release from the mitochondria [Packer & Murphy, 1994].

### **3. Classification of reactive oxygen species**

ROS generated in response to both endogenous and exogenous stimuli can be divided into Endogenous ROS and exogenous ROS. (Figure 1) [Ziech et al., 2010, Fukai & Nakamura, 2008, Klaunig & Kamendulis, 2004, Galaris et al., 2008].

Effect of Oxidative Stress on DNA Repairing Genes 53

In eukaryotic cells, aerobic respiration in the mitochondria is the main source for the generation of ROS. ROS are also produced by peroxisomal oxidation of fatty acids, microsomal cytochrome P450 metabolism of xenobiotic compounds, stimulation of phagocytosis by pathogens or lipopolysaccharides, arginine metabolism and tissue specific enzymes [Adly, 2010]. Enzymes in the cytosol, such as oxygenases, peroxidases and oxidases, generate small amounts of ROS. It is estimated that more than 95% of •O2<sup>−</sup> produced during normal metabolism are generated as a by-product from the electron transfer reactions at the inner mitochondrial membrane. Approximately 1-2% of the O2 consumed by mitochondria is converted into •O2− [Cadenas & Davies, 2000, Hashiguchi et al., 2004].Because of such a highly oxidative environment, mitochondria are also one of the main cellular targets of ROS induced damage, and in fact relatively high levels of oxidized proteins, lipids and nucleic acids are detected in mammalian mitochondria under normal

Another significant endogenous source of ROS production is via the reduction of molecular O2 by inflammatory cells [Klaunig & Kamendulis, 2004, Ziech et al., 2010, Fukai & Nakamura, 2008]. In this reaction, phagocytes produce •O2<sup>−</sup> via oxygen's monovalent reduction. In order to counterbalance ROS-mediated injury, endogenous antioxidant defense systems exist and function by quenching and clearing intracellular ROS activity and accumulation and maintaining redox equilibrium [Ziech et al., 2010, Dizdaroğlu et al., 1993]. Generated chronic oxidative stress damage to cellular macromolecules like DNA, lipids and proteins [Klaunig & Kamendulis, 2004, Ziech et al., 2010]. The endogenous enzymatic antioxidant defenses (SOD, GPx and catalase) can counterbalance oxidative microenvironments by chelating superoxide and various other peroxides. Also, the non-enzymatic endogenous antioxidants (Vitamins E and C, coenzyme Q, B-carotene and glutathione) have the ability to eliminate ROS activity. Thus, ROS can be considered as critical determinants of intracellular redox states and thus serve as important cellular regulatory mechanism(s) in both health and disease [Klaunig &

Environmental agents like radiation, xenobiotics and chlorinated compounds are significant inducers of cellular damage via ROS mediated toxicity [Galaris et al., 2008]. In addition, anticancer drugs, anesthetics, analgesics, trauma; radiation; electromagnetic fields; alcohol; cigarette smoke; medications; stress; allergens; cold, excessive exercise; dietary factors such as excess sugar, saturated fat and fried oils; malnutrition and various disease states have been considered as most established environmental sources of ROS [Cadenas & Davies, 2000]. Furthermore, exposure to ionizing and ultraviolet radiations also induces ROS-mediated alterations in major pathways associated with the control of cellular growth and survival. For instance, ultraviolet B rays can damage DNA directly and also increase ROS concentrations in epidermal cells [Mena et al., 2009]. In addition, urban air contains a mixture of oxidizing gases and particulates arising from a variety of sources, such as power plants, motor vehicles, wildfires and waste incinerators. Chronic exposure to polluted air induces irreversible damage to cellular macromolecules (DNA, proteins, lipids etc.) via ROS production and their accumulation in cells and tissues [Moller et al., 2008]. Metabolism of exogenous sources of ethanol and phenobarbital contributes to ROS generation through changes in the cytochrome P450 pathway [Wu &

metabolic conditions [Hashiguchi et al., 2004, Raha & Robinson, 2000].

**Endogenous ROS** 

Kamendulis, 2004, Ziech et al., 2010].

**Exogenous ROS** 

Cederbaum, 2003].

Fig. 1. Oxidative damage and repair of DNA.

#### **Endogenous ROS**

52 Selected Topics in DNA Repair

Fig. 1. Oxidative damage and repair of DNA.

In eukaryotic cells, aerobic respiration in the mitochondria is the main source for the generation of ROS. ROS are also produced by peroxisomal oxidation of fatty acids, microsomal cytochrome P450 metabolism of xenobiotic compounds, stimulation of phagocytosis by pathogens or lipopolysaccharides, arginine metabolism and tissue specific enzymes [Adly, 2010]. Enzymes in the cytosol, such as oxygenases, peroxidases and oxidases, generate small amounts of ROS. It is estimated that more than 95% of •O2<sup>−</sup> produced during normal metabolism are generated as a by-product from the electron transfer reactions at the inner mitochondrial membrane. Approximately 1-2% of the O2 consumed by mitochondria is converted into •O2− [Cadenas & Davies, 2000, Hashiguchi et al., 2004].Because of such a highly oxidative environment, mitochondria are also one of the main cellular targets of ROS induced damage, and in fact relatively high levels of oxidized proteins, lipids and nucleic acids are detected in mammalian mitochondria under normal metabolic conditions [Hashiguchi et al., 2004, Raha & Robinson, 2000].

Another significant endogenous source of ROS production is via the reduction of molecular O2 by inflammatory cells [Klaunig & Kamendulis, 2004, Ziech et al., 2010, Fukai & Nakamura, 2008]. In this reaction, phagocytes produce •O2<sup>−</sup> via oxygen's monovalent reduction. In order to counterbalance ROS-mediated injury, endogenous antioxidant defense systems exist and function by quenching and clearing intracellular ROS activity and accumulation and maintaining redox equilibrium [Ziech et al., 2010, Dizdaroğlu et al., 1993]. Generated chronic oxidative stress damage to cellular macromolecules like DNA, lipids and proteins [Klaunig & Kamendulis, 2004, Ziech et al., 2010]. The endogenous enzymatic antioxidant defenses (SOD, GPx and catalase) can counterbalance oxidative microenvironments by chelating superoxide and various other peroxides. Also, the non-enzymatic endogenous antioxidants (Vitamins E and C, coenzyme Q, B-carotene and glutathione) have the ability to eliminate ROS activity. Thus, ROS can be considered as critical determinants of intracellular redox states and thus serve as important cellular regulatory mechanism(s) in both health and disease [Klaunig & Kamendulis, 2004, Ziech et al., 2010].

#### **Exogenous ROS**

Environmental agents like radiation, xenobiotics and chlorinated compounds are significant inducers of cellular damage via ROS mediated toxicity [Galaris et al., 2008]. In addition, anticancer drugs, anesthetics, analgesics, trauma; radiation; electromagnetic fields; alcohol; cigarette smoke; medications; stress; allergens; cold, excessive exercise; dietary factors such as excess sugar, saturated fat and fried oils; malnutrition and various disease states have been considered as most established environmental sources of ROS [Cadenas & Davies, 2000]. Furthermore, exposure to ionizing and ultraviolet radiations also induces ROS-mediated alterations in major pathways associated with the control of cellular growth and survival. For instance, ultraviolet B rays can damage DNA directly and also increase ROS concentrations in epidermal cells [Mena et al., 2009]. In addition, urban air contains a mixture of oxidizing gases and particulates arising from a variety of sources, such as power plants, motor vehicles, wildfires and waste incinerators. Chronic exposure to polluted air induces irreversible damage to cellular macromolecules (DNA, proteins, lipids etc.) via ROS production and their accumulation in cells and tissues [Moller et al., 2008]. Metabolism of exogenous sources of ethanol and phenobarbital contributes to ROS generation through changes in the cytochrome P450 pathway [Wu & Cederbaum, 2003].

Effect of Oxidative Stress on DNA Repairing Genes 55

Decarboxylation of alloxan yields 5-hydroxyhydantoin (5-OH-Hyd) upon acidic treatment. Isodialuric acid is formed by deamination of 4-amino-6-hydroxy-2, 5(1H, 6H) pyrimidinedione. However, these two compounds may simultaneously exist in DNA as evidenced from the detection of their enol forms (5, 6-dihydroxycytosine (5, 6-diOH-Cyt) and 5, 6-dihydroxyuracil (5, 6-diOH-Ura), respectively) [11, 13]. 5-OH-6-hydroperoxide of cytosine undergoes intramolecular cyclisation to yield *trans*-1-carbamoyl-2-oxo-4, 5 dihydroxyimidazolidine as a major product in cytosine [Kohen & Nyska, 2002] However, this compound is formed as a minor product in DNA [Dizdaroglu et al., 1993a, Behrend et

Hydroxyl adduct radicals of guanine are formed by addition of •OH to the C4-, C5- and C8 positions of guanine, generating C4-OH-, C5-OH- and C8-OH-adduct radicals [O'Neill, 1983, Steenken, 1989, Candeisas & Steenken, 2000]. The 6-substituted purines such as adenine undergo analogous reactions, yielding at least two OH adducts, are formed: C4-OH and C8-OH adduct radicals [Steenken, 1989, Candeisas & Steenken, 2000]. C4-OH and C5- OH adduct radicals of purines differ in their redox properties, with C4-OH-adduct radicals

On the other hand, different mesomeric structures of these radicals may be oxidizing or reducing, a phenomenon called "redox ambivalence" [Vieira & Steenken, 1990]. Dehydration of C4-OH- and C5-OH-adduct radicals of purines reconstitutes the purine by first yielding a purine (–H)• radical, followed by reduction and protonation [Melvin et al., 1996]. The C4-OH-adduct radical of guanine also eliminates OH− to give rise to the guanine radical cation (guanine•+), which may deprotonate depending on pH to give guanine(–H)• [Moreno & Pryor, 1992]. Furthermore, on the basis of product analysis, the radical cation does not hydrate to lead to the C8-OH adduct radical and then to 8-hydroxyguanine by oxidation. However, it may react with 2'-deoxyribose in DNA by H abstraction, leading to

This diversity has been explained by the notion that the hydration of guanine•+ in ds-DNA may be much faster than that of monomeric guanine•+ [Candeias & Steenken, 2000]. O<sup>2</sup> readily reacts with guanine(–H)•; however, its reaction with the C4-OH-adduct radical of guanine is rather slow [Candeias & Steenken, 2000]. The reaction of guanine(-H)• with O<sup>2</sup> leads to imidazolone and oxazolone derivatives [Cadet et al., 1991]. However, this suggestion has not been confirmed by pulse radiolysis data and an alternative mechanism has been proposed [Melvin et al., 1996]. The C4-OH-adduct radical of adenine readily reacts with O2, however, final products of this reaction are not known [O'Neill & Chapman, 1985]. C8-OH adduct radicals of purines may be oxidized by oxidants including O2. In contrast to C4-OH

adduct radicals, their reaction with O2 is diffusion controlled [Vieira & Steenken, 1990].

8-Hydroxypurines are formed in DNA by the one-electron oxidation of C8-OH-adduct radicals [Bielski & Cabelli, 1995, Halliwell & Gutteridge, 1999] This reaction competes with the unimolecular opening of the imidazole ring by scission of the C8–N9 bond [Steenken, 1989, Candeias & Steenken, 2000]. The one-electron reduction of the ring-opened radical leads to 2, 6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from guanine and 4, 6 diamino-5-formamidopyrimidine (Fapy- Ade) from adenine [O'Neill, 1983, Breen & Murphy, 1995]. The C8-OH adduct radicals may also be reduced without ring opening to give rise to 7-hydro-8-hydroxypurines, which, as hemiorthoamides, are converted into formamidopyrimidines. However, 8-Hydroxypurines and formamidopyrimidines are unique in that they are formed in DNA both in the absence and presence of O2, although O2 increases the yields of 8-hydroxypurines. Moreover, other experimental conditions highly

being oxidising, and C5-OH- and C8-OH-adduct radicals being primarily reducing.

al., 1989, Dizdaroglu 1993b].

DNA strand breaks [O'Neill & Chapman, 1985].
