Cancer is a major health problem in almost all parts of the world. It could be resulted from both internal and environmental factors. These factors such as inherited mutations, inefficient immune system, smoking, bad diet,, infectious organisms, etc., are able to modulate our genes, especially tumor suppressor genes and DNA repair genes [8]. Cellular intermediates, along with unstable structures, affect cellular signaling pathways through transcription factors. These are nuclear factor-kappa B (NF-κB), signal transduction and transcription activator (STAT)-3, hypoxia inducible factor (HIF)-1α, kinases, various growth factors, cytokines and other proteins [56]. Extensive research showed that ROS has crucially important roles in modulating genes. Although low levels of ROS can be beneficial to cell, however, excessive accumulation of ROS could modify cell signaling pathways through the transcription factors [57]. Although ROS is balanced via endogenous antioxidant defense system, sometimes exogenous antioxidant needs to be supplied to counterbalance ROS-mediated injury. However, when oxidative status arises due to inefficient antioxidant system, chronic or cumulative oxidative stress eventually causes deleterious modifications in macromolecules such as protein, lipid and DNA [3]. ROS can also react with other cellular components such as phospholipids and proteins and result in the generation of secondary reactive intermediates and causes irreversible DNA bases by forming DNA adducts [58]. Formation of DNA adducts is the main step of carcinogenic process because, if such adducts cannot be repaired, they may lead to DNA damage and eventually to mutations [59]. Oxidative lesions play an important role in the etiology of cancer and (8-oxo-dG) lesions can be used as a critical biomarker for oxidative DNA damage [60, 61]. ROS can cause 8-OHdG in DNA and cause GC → TA transversions [62]. Therefore, 8-OHdG is widely used as biological markers of oxidative stress in studies of antioxidants and diseases associated with ROS [63]. Excess ROS levels cause DNA mutations such as GC → TA transversion mutations [64], single strand breaks and instability [65]. In human tumors, transversion from G to T is the most common mutation of the p53 repressor gene [66]. Excessive ROS levels can also increase carcinogenesis by inducing and sustaining oncogenic phenotypes of cancer cells [67, 68]. ROS are associated with three stages of carcinogenesis, initiation, promotion and progression. In cancer formation, ROS contributes to the initiation of nuclear or mitochondrial DNA mutations, including point mutations, deletions, chromosomal translocations, and others [69, 70]. The initiation stage transforms normal cells into cancer cells. Following the initiation stage, the initiated cells are expanded into colonies in the promotion stage, accompanied by cell proliferation and/or inhibition of apoptosis in this stage [7]. ROS promote the expansion of malignant cells by regulating cell proliferation/apoptosis-related genes and transcription factors such as nuclear factor-kappa B (NF-κB), activator protein-1 (AP-1), nuclear factor erythroid 2-related factor 2 (Nrf2), and hypoxia-inducible factor (HIF) [67, 71, 72]. Compared to normal cells, ROS levels have increased as a consequence of their metabolism in cancer cells, and they have a modified redox status to preserve malignant phenotypes [73].

oxide (NO) forming peroxynitrite (ONOO–) [44]. Another source of ROS is NOX localized in various parts of cellular membranes [45].ROS are also produced during the process of protein folding and disulfide bond formation in the endoplasmic reticulum. Glycoprotein endoplasmic reticulum oxidoreductin 1, protein disulfide isomerase and NOX4 are the main sources of ROS in the endoplasmic reticulum [46]. Under normal physiological conditions, the cells try to stabilize by eliminating the ROS production with the cleaning system [47]. Detoxification of ROS is facilitated by non-enzymatic molecules (e.g., Glutathione, flavonoids and vitamins A, C and

metal ions such as copper (Cu+2), zinc (Zn+2), manganese (Mn+2) or iron (Fe+2) as cofactors. "Different SOD enzymes are found in different compartments of the cell and are highly specific in regulating bound biologically bound processes" [48]. Catalase is an enzyme that facilitates the decomposition of hydrogen peroxide into water and the free oxygen molecule. The major localization of catalase in most eukaryotes is cytosol and peroxisomes [49]. Peroxiredoxins are thioredoxin peroxidases, which catalyze the reduction of hydrogen peroxide, organic hydroperoxides and peroxynitrite [50]. Glutathione has a significant role in cellular signaling and antioxidant defense system. It reacts directly with ROS and reactive nitrogen species (RNS) and is responsible for the detoxification of free radicals, membrane protection, metabolic regulation, modulation and signal transduction. The glutathione system involves reduced (GSH) and oxidized (GSSG) forms of glutathione. The enzymes required for the system includes glutathione reductase (GR), glutathione peroxidase (GPX), glutathione S-transferase (GST) [51]. Glutathione protects the cells against oxidative stress by reducing the disulfide bonds of cytoplasmic proteins to the cysteines. It is mostly synthesized in the cytosol of the cells and prevalent in most of the cells. It is then oxidized to glutathione disulfide. However, the oxidized form, GSSG, is predominant in endoplasmic reticulum. Glutathione peroxidase (GPx)

O2

alcohols [52]. Glutathione reductase converts GSSG to GSH [53]. Under physiological conditions, almost all of the glutathione are in reduced form because of a constitutive activity of glutathione reductase in cells, and the glutathione S-transferases (GSTs) are detoxifying enzymes that catalyze the ligation of various exogenous and endogenous electrophilic compounds [54]. GSTs are overexpressed in a variety of tumors to regulate MAPK pathways and

Normally, the human body naturally tries to compensate by producing endogenous or exogenously produced antioxidants against endogenously produced or exogenously taken oxidants. Endogenous and exogenous antioxidants act as "free radical scavengers" by preventing and repairing damages caused by ROS [7]. However, under oxidative stress conditions, excessive ROS can damage cellular proteins, lipids and DNA and cause deadly lesions

Cancer is a major health problem in almost all parts of the world. It could be resulted from both internal and environmental factors. These factors such as inherited mutations,

also play a role in the development of resistance to chemotherapeutics [55].

in the cells, which contribute to many human diseases, including cancer [56].

O2

and lipid peroxides to water and lipid

. SOD uses

E) or antioxidant enzymes that metabolize different ROS products.

24 Reactive Oxygen Species (ROS) in Living Cells

is an antioxidant enzyme that effectively reduces H2

**2.2. Role of reactive oxygen species in tumor formation**

SOD is a metalloenzyme that catalyzes the conversion of superoxide anion to H2

Compared to normal cells, cancer cells are loaded with more ROS. Therefore, they are more vulnerable to further ROS attack produced by exogenous ROS-generating factors (prooxidants) [74]. The role of ROS in cancer cells is described as "live by the sword, die by the sword" by Schumacher [75] or "a breath of life and death" by Fruehauf and Meyskens [76]. Therefore, prooxidant strategy should well be exploited to develop anticancer agents [77]. Although this strategy has not commonly been followed in conventional medicinal chemistry and is opposite to antioxidant therapy, however, it has promising sites that several ROS producing natural compounds such as phenethyl isothiocyanate [78], piperlongumine [79], curcumin [80] and parthenolide [81] are able to kill cancer cells efficiently. Recognizing the importance of ROS in cancer therapy, Jim Watson wrote: "The vast majority of all agents used kill ROS, directly or indirectly, cancer cells and produce ROS that inhibits important steps in the cell cycle" [74].

protection against cancer [91]. There have been several recent epidemiological studies that implicate dietary antioxidant phytochemicals such as carotenoids [92], phenolic compounds [93] and flavonoids [94] as protective agents against cancer and cardiovascular disease. Many studies have been conducted on how phytochemicals such as vitamin C and E, carotenoids,

Role of Antioxidant Phytochemicals in Prevention, Formation and Treatment of Cancer

http://dx.doi.org/10.5772/intechopen.72217

27

It is known that the concentration of antioxidant micronutrients such as vitamin C, vitamin E and carotenoids changes between high micromolar and low millimolar levels in human plasma and organs, while polyphenol concentrations are at high nanomolar to low micromolar levels [14]. However, despite their low levels, polyphenols have been reported to be more effective against oxidative stress than vitamin C [14]. In this respect, it has been suggested that phenolics are among the most active substances from natural sources, displaying a variety of health-promoting properties such as cytoprotective, antibacterial, antiviral, antiaging and anti-inflammatory effects [98, 99]. Some phytochemicals such as catechin and quercetin may show antioxidant effects not only due to molecular structures, but also by activating signaling

Although the antioxidant activity of phytochemicals is well recognized [98, 99, 101], they can also display prooxidant activities under certain conditions, such as at high doses or in the presence of metal ions [14, 102, 103]. Prooxidant or antioxidant activity has been shown to be dependent on the concentrations of phytochemicals and, in this context, studies using cell models have emphasized the prooxidative activities of polyphenols known as antioxidants such as quercetin, epicatechin and catechins containing epigallocatechin-3-gallate (EGCG) [14, 15, 104, 105]. For example, at high doses, quercetin (50 μM) has been shown to enhance

[104]. In another study, quercetin has been shown to reduce cell survival and viability, thiol content, total antioxidant capacity and SOD, CAT and glutathione transferase activity at higher concentrations (> 50 μM), while antioxidant activity of quercetin is observed only at low doses (0.1–20 μM) [15]. It has also been shown that flavonoids present in high concentrations can produce ROS with autoxidation (e.g., miksetin and quercetin) and redox cycling

In addition to the antioxidant concentration, prooxidant activity has been reported to be directly proportional to the total number of hydroxyl groups in a flavonoid molecule, and the presence of metal ions plays an important role [108]. Phytochemicals containing monoand dihydroxy-flavonoids showed no significant prooxidant activity, while compounds containing multiple hydroxyl groups, particularly in group B, have been shown to significantly increase hydroxyl radical production by the Fenton reaction [109, 110]. Galati et al. [16] found that EGCG was isolated in the presence of transition metals and caused oxidative damage to cellular DNA. In the presence of metal ions owing to their reducing capacity and forming chelates, antioxidants act directly on free radicals (−R.) by a scavenging process characterized by the donation of hydrogen atoms (resulting in the formation of –RH) or electrons (resulting formation of –R–) [111]. However, strong reducing power of antioxidants may have potential to affect metal ions such as Fe+3 and Cu+2, because they increase their ability to form highly

) in isolated mitochondria and cell culture medium

−

reactive HO**−.** radicals, originating from peroxides via Fenton's reaction [101, 112].

flavonoids and phenolic acids show anticarcinogenic effects [95–97].

pathways such as Nrf-2 [100].

(e.g., quercetin) [106, 107].

the production of superoxide radical (O2

#### **3. Phytochemicals**

#### **3.1. Phytochemicals and their presence in foods**

Phototherapy is the use of plant-based materials to prevent and treat diseases or to promote healthy life [82]. The word "phyto" comes from the Greek word for plant meaning, so phytochemicals mean plant chemicals. Phytochemicals are the bioactive non-nutrient plant chemicals found in fruits, vegetables, grains and other plant foods that have health-related effects. Vegetables and fruits consumed fresh or processed are the most important sources of phytochemicals necessary for human nutrition. Up to now, about 200,000 phytochemicals have been identified and 20,000 of them are derived from fruits, vegetables and grains [83].

Phytochemicals can be classified as carotenoids, phenolics, alkaloids, nitrogen-containing compounds and organosulphur compounds [84].These compounds are secondary metabolites with a variety of identifiable structures and are common features of the benzene ring and one or more hydroxyl groups. Generally, they are classified as flavonoids (anthocyanins, flavan-3-ols, flavonols, proanthocyanidins or flavones, non-hydrolyzable tannins, isoflavones and flavanones) and non-flavonoids (hydroxycinnamic, hydroxybenzoic acid, hydrolyzable tannins, benzoic acids and stilbenes) [85]. Phytochemicals are essential for the growth and reproduction of plants, and are produced as a response for defending plants against pathogens and stress in general [86]. In the last decade, the results of many researches have shown that phytochemicals have also a positive effect on human health. In general, phytochemicals, which are secondary metabolites found in plant foods such as alkaloids, phenolic compounds (flavonoids, isoflavonoids and anthocyanins) and terpenoids, have gained importance due to antioxidant, antiviral, antibacterial and anticancer effects [87]. Preclinical and clinical studies demonstrated that especially phenolic compounds have antimicrobial, anti-inflammatory, antioxidant, antiviral, anti-allergic, anticancer, anti-ulcer, antidiabetic, anti-plasmodia, antihypertensive and anticonvulsant effects [83]. In addition, food scientists and nutritionists think that consuming phytochemicals as part of a normal human diet is important for a healthy lifestyle [88].

#### **3.2. Phytochemicals and its antioxidant/prooxidant action**

A number phytochemicals, especially phenols and flavonoids, are found naturally in food and can behave like antioxidants [89]. Most plant foods contain phenols and flavonoids. Green leafy vegetables, fruits and yellow vegetables are especially rich in carotenoids, flavonoids and vitamin C vitamins. Vitamin C and vitamin E prevent the formation of carcinogenic nitrosamine [90]. Turmeric (*Curcuma domestica*), widely used in Indian food, contains a curcumin active ingredient, which is a strong antioxidant and a yellow coloring feature that can provide protection against cancer [91]. There have been several recent epidemiological studies that implicate dietary antioxidant phytochemicals such as carotenoids [92], phenolic compounds [93] and flavonoids [94] as protective agents against cancer and cardiovascular disease. Many studies have been conducted on how phytochemicals such as vitamin C and E, carotenoids, flavonoids and phenolic acids show anticarcinogenic effects [95–97].

compounds such as phenethyl isothiocyanate [78], piperlongumine [79], curcumin [80] and parthenolide [81] are able to kill cancer cells efficiently. Recognizing the importance of ROS in cancer therapy, Jim Watson wrote: "The vast majority of all agents used kill ROS, directly or indirectly, cancer cells and produce ROS that inhibits important steps in the cell cycle" [74].

Phototherapy is the use of plant-based materials to prevent and treat diseases or to promote healthy life [82]. The word "phyto" comes from the Greek word for plant meaning, so phytochemicals mean plant chemicals. Phytochemicals are the bioactive non-nutrient plant chemicals found in fruits, vegetables, grains and other plant foods that have health-related effects. Vegetables and fruits consumed fresh or processed are the most important sources of phytochemicals necessary for human nutrition. Up to now, about 200,000 phytochemicals have

Phytochemicals can be classified as carotenoids, phenolics, alkaloids, nitrogen-containing compounds and organosulphur compounds [84].These compounds are secondary metabolites with a variety of identifiable structures and are common features of the benzene ring and one or more hydroxyl groups. Generally, they are classified as flavonoids (anthocyanins, flavan-3-ols, flavonols, proanthocyanidins or flavones, non-hydrolyzable tannins, isoflavones and flavanones) and non-flavonoids (hydroxycinnamic, hydroxybenzoic acid, hydrolyzable tannins, benzoic acids and stilbenes) [85]. Phytochemicals are essential for the growth and reproduction of plants, and are produced as a response for defending plants against pathogens and stress in general [86]. In the last decade, the results of many researches have shown that phytochemicals have also a positive effect on human health. In general, phytochemicals, which are secondary metabolites found in plant foods such as alkaloids, phenolic compounds (flavonoids, isoflavonoids and anthocyanins) and terpenoids, have gained importance due to antioxidant, antiviral, antibacterial and anticancer effects [87]. Preclinical and clinical studies demonstrated that especially phenolic compounds have antimicrobial, anti-inflammatory, antioxidant, antiviral, anti-allergic, anticancer, anti-ulcer, antidiabetic, anti-plasmodia, antihypertensive and anticonvulsant effects [83]. In addition, food scientists and nutritionists think that consuming phyto-

been identified and 20,000 of them are derived from fruits, vegetables and grains [83].

chemicals as part of a normal human diet is important for a healthy lifestyle [88].

A number phytochemicals, especially phenols and flavonoids, are found naturally in food and can behave like antioxidants [89]. Most plant foods contain phenols and flavonoids. Green leafy vegetables, fruits and yellow vegetables are especially rich in carotenoids, flavonoids and vitamin C vitamins. Vitamin C and vitamin E prevent the formation of carcinogenic nitrosamine [90]. Turmeric (*Curcuma domestica*), widely used in Indian food, contains a curcumin active ingredient, which is a strong antioxidant and a yellow coloring feature that can provide

**3.2. Phytochemicals and its antioxidant/prooxidant action**

**3. Phytochemicals**

26 Reactive Oxygen Species (ROS) in Living Cells

**3.1. Phytochemicals and their presence in foods**

It is known that the concentration of antioxidant micronutrients such as vitamin C, vitamin E and carotenoids changes between high micromolar and low millimolar levels in human plasma and organs, while polyphenol concentrations are at high nanomolar to low micromolar levels [14]. However, despite their low levels, polyphenols have been reported to be more effective against oxidative stress than vitamin C [14]. In this respect, it has been suggested that phenolics are among the most active substances from natural sources, displaying a variety of health-promoting properties such as cytoprotective, antibacterial, antiviral, antiaging and anti-inflammatory effects [98, 99]. Some phytochemicals such as catechin and quercetin may show antioxidant effects not only due to molecular structures, but also by activating signaling pathways such as Nrf-2 [100].

Although the antioxidant activity of phytochemicals is well recognized [98, 99, 101], they can also display prooxidant activities under certain conditions, such as at high doses or in the presence of metal ions [14, 102, 103]. Prooxidant or antioxidant activity has been shown to be dependent on the concentrations of phytochemicals and, in this context, studies using cell models have emphasized the prooxidative activities of polyphenols known as antioxidants such as quercetin, epicatechin and catechins containing epigallocatechin-3-gallate (EGCG) [14, 15, 104, 105]. For example, at high doses, quercetin (50 μM) has been shown to enhance the production of superoxide radical (O2 − ) in isolated mitochondria and cell culture medium [104]. In another study, quercetin has been shown to reduce cell survival and viability, thiol content, total antioxidant capacity and SOD, CAT and glutathione transferase activity at higher concentrations (> 50 μM), while antioxidant activity of quercetin is observed only at low doses (0.1–20 μM) [15]. It has also been shown that flavonoids present in high concentrations can produce ROS with autoxidation (e.g., miksetin and quercetin) and redox cycling (e.g., quercetin) [106, 107].

In addition to the antioxidant concentration, prooxidant activity has been reported to be directly proportional to the total number of hydroxyl groups in a flavonoid molecule, and the presence of metal ions plays an important role [108]. Phytochemicals containing monoand dihydroxy-flavonoids showed no significant prooxidant activity, while compounds containing multiple hydroxyl groups, particularly in group B, have been shown to significantly increase hydroxyl radical production by the Fenton reaction [109, 110]. Galati et al. [16] found that EGCG was isolated in the presence of transition metals and caused oxidative damage to cellular DNA. In the presence of metal ions owing to their reducing capacity and forming chelates, antioxidants act directly on free radicals (−R.) by a scavenging process characterized by the donation of hydrogen atoms (resulting in the formation of –RH) or electrons (resulting formation of –R–) [111]. However, strong reducing power of antioxidants may have potential to affect metal ions such as Fe+3 and Cu+2, because they increase their ability to form highly reactive HO**−.** radicals, originating from peroxides via Fenton's reaction [101, 112].

$$\text{Antioxidant (AH)} + \text{Fe}^{3+} \text{(or } \text{Cu}^{2+}\text{)} \rightarrow \text{A}^{\cdot} + \text{Fe}^{2+} \text{(or } \text{Cu}^{\cdot}\text{)} + \text{H}^{\cdot} \tag{1}$$

$$\mathrm{H\_2O\_2 + Fe^{2+} (or Cu+) \to HO^\cdot + HO^- + Fe^{3+} (or Cu^{2+})}\tag{2}$$

$$\rm Fe^{2+} + \rm O\_{2} + H\_{2}O \rightarrow Fe^{3+} + HO\_{2} + HO^{-} \tag{3}$$

the human mononuclear leukocyte cells [120]. Oxidases such as lipoxygenase (LO), myeloperoxidase (MPO), NADPH oxidase and xanthine oxidase (XO) are considered to be one of the important mechanisms by which phytochemicals inhibits the formation of high amounts of ROS [119]. Phytochemicals also inhibit enzymes indirectly involved in the oxidative process, such as phospholipase A2 (FLA2), by stimulating known antioxidant enzyme activities such as catalase and superoxide dismutase (SOD) [121]. For this reason, flavonoids can be considered as phytochemicals that can interfere directly or indirectly with the formation of free radicals

Role of Antioxidant Phytochemicals in Prevention, Formation and Treatment of Cancer

http://dx.doi.org/10.5772/intechopen.72217

29

It appears that antioxidants are found in the body at sufficient concentrations to prevent accumulation of prooxidants (oxidative stress state) and that exogenous antioxidants play an important role in maintaining healthy biological systems and establishing redox hemostasis at physiological (nutritional) doses [123]. However, exogenous antioxidants, particularly phenolic compounds, can participate in redox reactions that can function as antioxidants (electron donors) or prooxidants (electron acceptors), depending on their environment [14, 103]. The antioxidant or prooxidant activity also depends on their concentration [98]. It is now assumed that exogenous antioxidants, including polyphenols, act as "double edged swords" according to their cellular redox status [123]. Yordi and Pérez [124] recently published a list of such compounds and their dietary sources. Some of the most abundant flavonoids and phenolic acids found in plants were reported to act as prooxidants, besides antioxidant activities: quercetin, curcumin, mycetin, kaempferol and caffeic, chlorogenic, ferulic acids and phenolic acids were also demonstrated as prooxidants [125–129]. Several studies have shown that oxidative stress is either mediated through the formation of ROS or inhibition of antioxidant systems from prooxidant agents [130]. For this reason, the type, phylogenetic, and matrix of phytochemicals may be determining factors affecting the balance between beneficial or deleterious effects of these natural compounds [123]. It is known that the development of many chronic diseases may be due to an oxidative stress, which antioxidant/pro-oxidant balance cannot provide and may lead to a pathological process [131]. The prooxidants catalyze the oxidative reactions of biomolecules, which may lead to cellular dysfunction [132]. It was demonstrated that increased prooxidant activity had damaged to biomolecules such as DNA, proteins and lipids that were able to lead to a variety of diseases such as cancer and cellular death [98, 132]. It was firstly demonstrated that resveratrol can induce oxidative DNA damage in the presence of copper ions [133]. Although it is associated with consumption of hot tea, it has been shown that too much tea consumption (>1 l/d) is associated with an increase in the incidence of esophageal cancer in some countries such as northern Iran or India [20]. At

O2

of this, taking plants with high dose phytochemicals is not always effective or safe, sometimes

In the use of phytochemicals, it is necessary to distinguish pharmacological doses from physiological (nutritional) dosages. Clinically, physiological doses are usually used to optimize or maintain optimal health. The pharmacological dose generally requires a doctor's prescription

in the mouth cavity [132]. Because

[122].

**4.2. The role of phytochemicals in cancer formation**

the same time, green tea has been shown to produce H2

may have toxic effects.

$$\text{Fe}^{2+} + \text{HO}\_{2}^{\cdot -} + \text{H}\_{2}\text{O} \rightarrow \text{Fe}^{3+} \text{HO}^{-} + \text{H}\_{2}\text{O}\_{2} \tag{4}$$

The antioxidant phenolic compounds, when scavenging the free radicals, can form less reactive phenoxyl radicals and are stabilized by delocalization of unpaired electrons around the aromatic ring [113]. However, even though these radicals are relatively stable, they may also show prooxidant activities [16]. However, it should be emphasized that natural compounds may have harmful effects as well as beneficial effects (independent of their anti-oxidative properties); for example, inflammation processes, activation of certain cellular pathways such as nitrogen and dicarbonyl metabolisms [16, 114]. It has been demonstrated that β-carotene at low doses exhibited antioxidant [115] and anti-inflammatory [116] properties in human HL-60 cells. However, at high doses, it exhibited prooxidant activity [115] and pro-inflammatory effects [116].

#### **4. Chemopreventive role of phytochemicals in cancer**

#### **4.1. The role of phytochemicals in cancer prevention**

Epidemiological studies have shown that the consumption of fruits and vegetables regularly reduces the risk of developing chronic diseases such as cancer and cardiovascular diseases [117]. The data suggest that people fed on an antioxidant-rich diet have a higher risk of chronic diseases and mortality than those who consume less fruits and vegetables. In a cohort study, Serafini [118] suggest that high intake of antioxidant-rich fresh fruits, root vegetables and vegetables is associated with a reduction in mortality and antioxidant-rich nutraceuticals have a protective effect on cancer development.

While the biological functions of polyphenols and/or metabolism in the human body are not completely known, there is consensus that antioxidant activity of flavonoids may be a combination of metal chelating and free radical scavenging properties [118]. Therefore, the structure of polyphenols enables them having free radical scavenging activity. Degree of methoxylation and the number of hydroxyl groups are important factors enabling them to have antioxidant properties. As for phenolic acids, inhibition of oxidation is associated with the cleavage of alkoxyl and peroxyl radicals, the cleavage of metal ions by the orthodihydroxy phenolic structure, and the production of α-tocopherol by reduction of tocopheryl radical [119]. Recently, we have shown that the naringenin-oxime compound, having one or more hydroxyl groups than naringenin, had more antioxidant and anti-genotoxic potentials than the naringenin in the human mononuclear leukocyte cells [120]. Oxidases such as lipoxygenase (LO), myeloperoxidase (MPO), NADPH oxidase and xanthine oxidase (XO) are considered to be one of the important mechanisms by which phytochemicals inhibits the formation of high amounts of ROS [119]. Phytochemicals also inhibit enzymes indirectly involved in the oxidative process, such as phospholipase A2 (FLA2), by stimulating known antioxidant enzyme activities such as catalase and superoxide dismutase (SOD) [121]. For this reason, flavonoids can be considered as phytochemicals that can interfere directly or indirectly with the formation of free radicals [122].

#### **4.2. The role of phytochemicals in cancer formation**

Antioxidant (AH) + Fe3+ (or Cu2+) → A**.** + Fe2+ (or Cu+) + H+ (1)

H2 O2 + Fe2+ (or Cu+) → HO**.** + HO− + Fe3+ (or Cu2+) (2)

The antioxidant phenolic compounds, when scavenging the free radicals, can form less reactive phenoxyl radicals and are stabilized by delocalization of unpaired electrons around the aromatic ring [113]. However, even though these radicals are relatively stable, they may also show prooxidant activities [16]. However, it should be emphasized that natural compounds may have harmful effects as well as beneficial effects (independent of their anti-oxidative properties); for example, inflammation processes, activation of certain cellular pathways such as nitrogen and dicarbonyl metabolisms [16, 114]. It has been demonstrated that β-carotene at low doses exhibited antioxidant [115] and anti-inflammatory [116] properties in human HL-60 cells. However, at high doses, it exhibited prooxidant activity [115] and pro-inflam-

Epidemiological studies have shown that the consumption of fruits and vegetables regularly reduces the risk of developing chronic diseases such as cancer and cardiovascular diseases [117]. The data suggest that people fed on an antioxidant-rich diet have a higher risk of chronic diseases and mortality than those who consume less fruits and vegetables. In a cohort study, Serafini [118] suggest that high intake of antioxidant-rich fresh fruits, root vegetables and vegetables is associated with a reduction in mortality and antioxidant-rich nutraceuticals

While the biological functions of polyphenols and/or metabolism in the human body are not completely known, there is consensus that antioxidant activity of flavonoids may be a combination of metal chelating and free radical scavenging properties [118]. Therefore, the structure of polyphenols enables them having free radical scavenging activity. Degree of methoxylation and the number of hydroxyl groups are important factors enabling them to have antioxidant properties. As for phenolic acids, inhibition of oxidation is associated with the cleavage of alkoxyl and peroxyl radicals, the cleavage of metal ions by the orthodihydroxy phenolic structure, and the production of α-tocopherol by reduction of tocopheryl radical [119]. Recently, we have shown that the naringenin-oxime compound, having one or more hydroxyl groups than naringenin, had more antioxidant and anti-genotoxic potentials than the naringenin in

**.** + HO− (3)

**.** + H2 O → Fe3+ HO− + H2 O2 (4)

Fe2+ + O2 + H2 O → Fe3+ + HO2

**4. Chemopreventive role of phytochemicals in cancer**

**4.1. The role of phytochemicals in cancer prevention**

have a protective effect on cancer development.

Fe2+ + HO2

28 Reactive Oxygen Species (ROS) in Living Cells

matory effects [116].

It appears that antioxidants are found in the body at sufficient concentrations to prevent accumulation of prooxidants (oxidative stress state) and that exogenous antioxidants play an important role in maintaining healthy biological systems and establishing redox hemostasis at physiological (nutritional) doses [123]. However, exogenous antioxidants, particularly phenolic compounds, can participate in redox reactions that can function as antioxidants (electron donors) or prooxidants (electron acceptors), depending on their environment [14, 103]. The antioxidant or prooxidant activity also depends on their concentration [98]. It is now assumed that exogenous antioxidants, including polyphenols, act as "double edged swords" according to their cellular redox status [123]. Yordi and Pérez [124] recently published a list of such compounds and their dietary sources. Some of the most abundant flavonoids and phenolic acids found in plants were reported to act as prooxidants, besides antioxidant activities: quercetin, curcumin, mycetin, kaempferol and caffeic, chlorogenic, ferulic acids and phenolic acids were also demonstrated as prooxidants [125–129]. Several studies have shown that oxidative stress is either mediated through the formation of ROS or inhibition of antioxidant systems from prooxidant agents [130]. For this reason, the type, phylogenetic, and matrix of phytochemicals may be determining factors affecting the balance between beneficial or deleterious effects of these natural compounds [123]. It is known that the development of many chronic diseases may be due to an oxidative stress, which antioxidant/pro-oxidant balance cannot provide and may lead to a pathological process [131]. The prooxidants catalyze the oxidative reactions of biomolecules, which may lead to cellular dysfunction [132]. It was demonstrated that increased prooxidant activity had damaged to biomolecules such as DNA, proteins and lipids that were able to lead to a variety of diseases such as cancer and cellular death [98, 132]. It was firstly demonstrated that resveratrol can induce oxidative DNA damage in the presence of copper ions [133]. Although it is associated with consumption of hot tea, it has been shown that too much tea consumption (>1 l/d) is associated with an increase in the incidence of esophageal cancer in some countries such as northern Iran or India [20]. At the same time, green tea has been shown to produce H2 O2 in the mouth cavity [132]. Because of this, taking plants with high dose phytochemicals is not always effective or safe, sometimes may have toxic effects.

In the use of phytochemicals, it is necessary to distinguish pharmacological doses from physiological (nutritional) dosages. Clinically, physiological doses are usually used to optimize or maintain optimal health. The pharmacological dose generally requires a doctor's prescription to treat the specific disease, because in the intake of antioxidant micronutrients, pharmacological doses are not equal to physiological doses and the intake of antioxidant micronutrients may be toxic and can generate cancer.

survival or apoptotic signal is related to the dose, duration, type and location of ROS production. In short, while moderate levels of ROS are required for cancer cells to survive, extreme

Role of Antioxidant Phytochemicals in Prevention, Formation and Treatment of Cancer

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31

Although phytochemicals of plant origin are known to have preventive effects on cancer and they are widely used in developed countries [151], numerous studies have revealed that many of these agents can kill cancer cells [56]. Some drugs, nowadays, used in chemotherapy are natural plant-derived products such as (e.g., paclitaxel, vincristine, vinblastine, bleomycin, mitomycin, doxorubicin, idarubicin, aclarubicin and actinomycin D). Other chemicals such as curcumin, epigallocatechin-3-gallate, genistein, resveratrol, camptothecin, perillyl alcohol, lycopene, phenylethyl isothiocyanate, sulforaphane, aplidin, eicosapentaenoic acid, linoleic acid, ursodeoxycholic acid, and vitamin C are in the clinical test stage for the treatment of cancer [1].

The potentials of some phytochemicals to treat cancer were evidenced by both *in vitro* cell culture systems and *in vivo* mice models [152, 153]. However, their therapeutic effect on cancer cells has not been elucidated yet. There are several mechanisms offered for the cytotoxicity of phytochemicals including the inhibition of topoisomerases, kinases and prooxidant actions [16]. Although many phytochemicals known as antioxidants can protect the cell from the oxidative stress and neutralize the damaging effect of ROS, however, they can, on the other hand, be cytotoxic at high concentrations. However, the mechanism of dual protectivedestructive behavior of flavonoids is not exactly known. It is highly possible that the prooxidant effect is responsible for the selective antiproliferative activity of these compounds, and ROS are key signaling molecules to modulate cell death [154]. Because, many phytochemical agents exhibit prooxidant action, particularly in the presence of transition metal ions such as copper [13, 155]. The prooxidant activity of individual dietary polyphenols and their ability to induce mitochondrial dysfunction and consequently apoptosis has been suggested a possible anticancer mechanism [136]. There seems to be enough evidence to support phytochemicals-mediated production of ROS, a prooxidant action that is responsible for their ability to

induce apoptosis in cancer cells. It was observed that the accumulation of H2

paclitaxel-induced cancer cell death both *in vitro* and *in vivo* [156, 157]. Being well known

can induce selective killing of cancer cells, it seems possible that paclitaxel induced

 production plays a role in the selective anticancer effects of this natural product [158]. Several *in vitro* and *in vivo* studies have also shown that phytochemicals such as catechins, phisapubesin B, daucosterol and hesperetin can induce apoptosis induction in various cancer cells and animal models [33, 159–162]. Recently, we also demonstrated cytotoxic, apoptotic, ROS generating and DNA damaging effects of naringenin on cancer and normal cells *in vitro* in cell culture medium [163]. In the same line, the apoptosis inducing effect of EGCG has been shown to be due to an increase in caspase-3, -9 and -8 [76–79] expression [164]. Similarly, the intrinsic pathway (FAS-independent, caspase 8-independent) by down regulation of ellagic acid Bcl-xL and release of cytochrome C, and colon cancer induced apoptosis in Caco-2 cells [165]. Interestingly, ellagic acid, quercetin and curcumin were found to induce ROS formation, DNA damage and apoptosis synergistically in cancer cell lines [132, 166, 167]. Recently, a combination of chemotherapeutic drug imatinib and curcumin has been used in a cancer patient and has been shown to increase efficacy in cancer treatment [168].  Role of phytochemicals in cancer therapy**

One of the main features of cancer cells is the survival ability. For this reason, the main goal of cancer therapy is to kill cancer cells by selecting them without harming normal cells. There are various therapeutic methods to treat cancer including chemotherapy, radiotherapy, and/ or surgery. Chemotherapy is one of the basic modalities in the treatment of cancer patients [110]. Although chemotherapy is aimed at removing the desired primary target tumor cells, normal cells are also affected and produce many side-effects in multiple organ systems [26, 134, 135]. For this reason, efforts are being made to develop alternative and effective treatment methods. The studies have focused on the active components of plants with low toxicity and high selectivity for killing cancer cells.

The primary mechanism of many chemotherapeutic drugs against cancer cells is the formation of ROS or free radicals [26, 27]. Indeed, there is a realistic approach to treatments aimed at strikingly increasing intracellular ROS to kill cancer cells by reducing antioxidant capacity [136]. This can be achieved by using compounds that inhibit antioxidant systems or by inhibiting specific signaling pathways that upregulate antioxidants in cancer cells. The resulting increase in ROS can stimulate tumor cell death through harmful functions of ROS, or through apoptosis-specific induction of death signaling pathways. Chemotherapeutics that makeup ROS include alkylating agents (melfalan, cyclophosphamide), anthracyclines (doxorubicin, epirubicin), podophyllin derivatives (etoposide), platinum coordination complexes (cisplatin, carboplatin) and camptothecin (topokan, irinotecan) [137]. Because, high ROS levels results in acute damage to cellular components such as DNA, proteins and lipids. ROS can attack DNA due to its strong reactivity and can cause DNA base oxidation, DNA lesion and damage to the DNA helix [138]. One of the first drugs developed based on ROS production characteristics was the procarbazine [139]. It hydrolyzes in aqueous solutions and the cytotoxic effects of the drug are the result of H2 O2 production [140]. Similar to chemotherapy, radiotherapy also kills cancer cells by producing ROS [141]. Cancer cells can be killed by three pathways: apoptosis, necrosis, and autophagy [142–144]. Apoptosis is a tightly regulated form of cell death and can be initiated by death receptors (extrinsic pathways) or mitochondria (internal pathways), and both extrinsic and intrinsic pathways of apoptosis are associated with ROS [145]. ROS is also required for Fas phosphorylation at the tyrosine residue, a signal for Fas-associated death pathway and caspase-8 and for apoptosis induction [146]. Although it is not known that ROS induces apoptosis in excessive amounts, high levels may cause necrotic cell death. In some cases, ROS can trigger both apoptosis and necrosis in cancer cells. For example, it has been determined that low H2 O2 concentrations in Jurkat T lymphocytes cause apoptosis by caspase activation in cells, while higher concentrations cause cell death by inducing necrosis [147]. Studies have also shown the role of ROS as a signaling molecule in the stimulation of autophagic cell death in cancer cells [148]. Besides the ability of cells to kill, ROS is also necessary for the survival of cancer cells. In fact, the ability of cancer cells to differentiate ROS as a survival or apoptotic signal is related to the dose, duration, type and location of ROS production. In short, while moderate levels of ROS are required for cancer cells to survive, extreme levels kill them [149, 150].

to treat the specific disease, because in the intake of antioxidant micronutrients, pharmacological doses are not equal to physiological doses and the intake of antioxidant micronutrients

One of the main features of cancer cells is the survival ability. For this reason, the main goal of cancer therapy is to kill cancer cells by selecting them without harming normal cells. There are various therapeutic methods to treat cancer including chemotherapy, radiotherapy, and/ or surgery. Chemotherapy is one of the basic modalities in the treatment of cancer patients [110]. Although chemotherapy is aimed at removing the desired primary target tumor cells, normal cells are also affected and produce many side-effects in multiple organ systems [26, 134, 135]. For this reason, efforts are being made to develop alternative and effective treatment methods. The studies have focused on the active components of plants with low toxicity and

The primary mechanism of many chemotherapeutic drugs against cancer cells is the formation of ROS or free radicals [26, 27]. Indeed, there is a realistic approach to treatments aimed at strikingly increasing intracellular ROS to kill cancer cells by reducing antioxidant capacity [136]. This can be achieved by using compounds that inhibit antioxidant systems or by inhibiting specific signaling pathways that upregulate antioxidants in cancer cells. The resulting increase in ROS can stimulate tumor cell death through harmful functions of ROS, or through apoptosis-specific induction of death signaling pathways. Chemotherapeutics that makeup ROS include alkylating agents (melfalan, cyclophosphamide), anthracyclines (doxorubicin, epirubicin), podophyllin derivatives (etoposide), platinum coordination complexes (cisplatin, carboplatin) and camptothecin (topokan, irinotecan) [137]. Because, high ROS levels results in acute damage to cellular components such as DNA, proteins and lipids. ROS can attack DNA due to its strong reactivity and can cause DNA base oxidation, DNA lesion and damage to the DNA helix [138]. One of the first drugs developed based on ROS production characteristics was the procarbazine [139]. It hydrolyzes in aqueous solutions and the cytotoxic effects of the

cancer cells by producing ROS [141]. Cancer cells can be killed by three pathways: apoptosis, necrosis, and autophagy [142–144]. Apoptosis is a tightly regulated form of cell death and can be initiated by death receptors (extrinsic pathways) or mitochondria (internal pathways), and both extrinsic and intrinsic pathways of apoptosis are associated with ROS [145]. ROS is also required for Fas phosphorylation at the tyrosine residue, a signal for Fas-associated death pathway and caspase-8 and for apoptosis induction [146]. Although it is not known that ROS induces apoptosis in excessive amounts, high levels may cause necrotic cell death. In some cases, ROS can trigger both apoptosis and necrosis in cancer cells. For example, it has

caspase activation in cells, while higher concentrations cause cell death by inducing necrosis [147]. Studies have also shown the role of ROS as a signaling molecule in the stimulation of autophagic cell death in cancer cells [148]. Besides the ability of cells to kill, ROS is also necessary for the survival of cancer cells. In fact, the ability of cancer cells to differentiate ROS as a

production [140]. Similar to chemotherapy, radiotherapy also kills

concentrations in Jurkat T lymphocytes cause apoptosis by

may be toxic and can generate cancer.

30 Reactive Oxygen Species (ROS) in Living Cells

high selectivity for killing cancer cells.

drug are the result of H2

been determined that low H2

O2

O2

**4.3. Role of phytochemicals in cancer therapy**

Although phytochemicals of plant origin are known to have preventive effects on cancer and they are widely used in developed countries [151], numerous studies have revealed that many of these agents can kill cancer cells [56]. Some drugs, nowadays, used in chemotherapy are natural plant-derived products such as (e.g., paclitaxel, vincristine, vinblastine, bleomycin, mitomycin, doxorubicin, idarubicin, aclarubicin and actinomycin D). Other chemicals such as curcumin, epigallocatechin-3-gallate, genistein, resveratrol, camptothecin, perillyl alcohol, lycopene, phenylethyl isothiocyanate, sulforaphane, aplidin, eicosapentaenoic acid, linoleic acid, ursodeoxycholic acid, and vitamin C are in the clinical test stage for the treatment of cancer [1].

The potentials of some phytochemicals to treat cancer were evidenced by both *in vitro* cell culture systems and *in vivo* mice models [152, 153]. However, their therapeutic effect on cancer cells has not been elucidated yet. There are several mechanisms offered for the cytotoxicity of phytochemicals including the inhibition of topoisomerases, kinases and prooxidant actions [16]. Although many phytochemicals known as antioxidants can protect the cell from the oxidative stress and neutralize the damaging effect of ROS, however, they can, on the other hand, be cytotoxic at high concentrations. However, the mechanism of dual protectivedestructive behavior of flavonoids is not exactly known. It is highly possible that the prooxidant effect is responsible for the selective antiproliferative activity of these compounds, and ROS are key signaling molecules to modulate cell death [154]. Because, many phytochemical agents exhibit prooxidant action, particularly in the presence of transition metal ions such as copper [13, 155]. The prooxidant activity of individual dietary polyphenols and their ability to induce mitochondrial dysfunction and consequently apoptosis has been suggested a possible anticancer mechanism [136]. There seems to be enough evidence to support phytochemicals-mediated production of ROS, a prooxidant action that is responsible for their ability to induce apoptosis in cancer cells. It was observed that the accumulation of H2 O2 is crucial for paclitaxel-induced cancer cell death both *in vitro* and *in vivo* [156, 157]. Being well known that H2 O2 can induce selective killing of cancer cells, it seems possible that paclitaxel induced H2 O2 production plays a role in the selective anticancer effects of this natural product [158]. Several *in vitro* and *in vivo* studies have also shown that phytochemicals such as catechins, phisapubesin B, daucosterol and hesperetin can induce apoptosis induction in various cancer cells and animal models [33, 159–162]. Recently, we also demonstrated cytotoxic, apoptotic, ROS generating and DNA damaging effects of naringenin on cancer and normal cells *in vitro* in cell culture medium [163]. In the same line, the apoptosis inducing effect of EGCG has been shown to be due to an increase in caspase-3, -9 and -8 [76–79] expression [164]. Similarly, the intrinsic pathway (FAS-independent, caspase 8-independent) by down regulation of ellagic acid Bcl-xL and release of cytochrome C, and colon cancer induced apoptosis in Caco-2 cells [165]. Interestingly, ellagic acid, quercetin and curcumin were found to induce ROS formation, DNA damage and apoptosis synergistically in cancer cell lines [132, 166, 167]. Recently, a combination of chemotherapeutic drug imatinib and curcumin has been used in a cancer patient and has been shown to increase efficacy in cancer treatment [168]. In addition, pharmacological doses of vitamin C and curcumin have been reported to be used in the treatment of different types of cancer, and successful results have been reported [169–174].

knowledge of the oxidative status of the applied organism are vital to the phytochemical agent used in the prevention or treatment of cancer. Otherwise, treatment with phytochemi-

1 Department of Medical Biochemistry, Faculty of Medicine, Bezmialem Vakif University,

2 Department of Plant Protection, Faculty of Agriculture, Harran University, Sanliurfa,

[1] Boyle P, Levin B. 2008 World Cancer Report 2008. International Agency for Research on

[2] Sadikovic B, Al-Romaih K, Squire J, Zielenska M. Cause and consequences of genetic and epigenetic alterations in human cancer. Current Genomics. 2008;**9**(6):394-408

[3] Ziech D, Franco R, Georgakilas AG, Georgakila S, Malamou-Mitsi V, Schoneveld O, et al. The role of reactive oxygen species and oxidative stress in environmental carcinogenesis and biomarker development. Chemico-Biological Interactions. 2010;**188**(2):334-339

[4] Waris G, Ahsan H. Reactive oxygen species: Role in the development of cancer and vari-

[5] Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: The metabolic requirements of cell proliferation. Science. 2009;**324**(5930):1029-1033

[6] Klaunig JE, Kamendulis LM. The role of oxidative stress in carcinogenesis. Annual

[7] Valko M, Rhodes C, Moncol J, Izakovic M, Mazur M.Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chemico-Biological Interactions. 2006;**160**(1):1-40

[8] Halliwell B. Oxidative stress and cancer: Have we moved forward? Biochemical Journal.

[9] Aniagu SO. Toxicological evaluation of herbal medicines: Approaches and perspectives. In: Evaluation of Herbal Medicinal Products. Pharmaceutical Press; 2009. pp. 444-446.

ous chronic conditions. Journal of Carcinogenesis. 2006;**5**(1):14

Review of Pharmacology and Toxicology. 2004;**44**:239-267

and Murat Dikilitas2

Role of Antioxidant Phytochemicals in Prevention, Formation and Treatment of Cancer

http://dx.doi.org/10.5772/intechopen.72217

33

cals may result in an opposite result to the desired effect.

\*, Eray Metin Guler1

\*Address all correspondence to<EMAIL_ADDRESS>
Cancer. Lyon/Geneva: WHO Press; 2008

**Author details**

Istanbul, Turkey

**References**

2007;**401**(1):1-11

(Chapter 30)

Turkey

Abdurrahim Kocyigit1

Studies have shown that many phytochemicals show more cytotoxic effects in various cancer cells than in normal cells [175–177]. Some studies have shown that polyphenols such as EGCG and genistein kill cancer cells more at the same dose with apoptosis than normal cells [178, 179]. We also showed that an herbal medicine named ankaferd, derived from different plant extracts, killed cancer cells more than normal cells at the same doses [180]. However, it is not clear how this differential effect occurs. This may be due to the difference in metabolism between cancer cells and normal cells. Since the metabolism of cancer cells is higher than normal cells, endogenous ROS production levels are much higher than normal cells [181]. The use of prooxidant phytochemicals emerges as an exciting strategy to target tumor cells selectively, due to further increase in ROS levels in cancer cells. Indeed, our findings show that the production of ROS in cancer cells to which phytochemicals are applied is significantly higher than normal cells and that there is a positive correlation between ROS level and cell death [163, 180]. The advantage of such a strategy is that it is not significantly affected by the fact that basal ROS levels of normal cells are lower than cancer cells and therefore less dependent on antioxidants.

Although many *in vitro* cell culture studies have been carried out on the use of phytochemicals in the treatment of cancer, the number of experimental animals and clinical trials *in vivo* is low. An important problem in *in vivo* studies is that phytochemicals are digestion, absorption and bioavailability. Bioavailability is very low due to low absorption rate of many phytochemicals [182]. Therefore, enteral administration is preferred for cancer treatment [168, 171].

#### **5. Conclusion**

In conclusion, extensive researches over the past half a century have indicated that oxygen ROS play an important role in cancer metabolism. ROS are one of the main components of cell signaling pathways and have been shown to take roles in regulating cell transformation, survival, proliferation, invasion, angiogenesis, and metastasis [73, 168]. On the other hand, ROS can also suppress tumor progression. However, most chemotherapeutic and radiotherapeutic agents are designated to reduce the impact of ROS by augmenting ROS stress in cancer cells [183]. Due to these dual roles of ROS, both prooxidant-based and antioxidant-based anticancer agents have been developed [184]. It is clear that numerous chemotherapeutics mediate their effects by inducing ROS generation. However, unwarranted side effects of synthetic anticancer drugs should be minimized in healthy cells. For this reason, researchers continue to look at the nature and explore the potential for cancer treatment. A great number of phytochemicals including some of the vitamins, flavonoids, terpenoids, carotenoids, phenolics, phytoestrogens, minerals and antioxidants in plant materials are used for chemoprevention of cancer. However, various *in vitro* and *in vivo* experiments have shown that phytochemicals have carcinogenic potential as well as protective and curative effects against cancer. Many studies have shown that these three different effects of phytochemicals on cancer are related to the molecular structure of phytochemicals, bioavailability, dose and the oxidative status of the administered organism. For this reason, the molecular structure, bioavailability and knowledge of the oxidative status of the applied organism are vital to the phytochemical agent used in the prevention or treatment of cancer. Otherwise, treatment with phytochemicals may result in an opposite result to the desired effect.

#### **Author details**

pharmacological doses of vitamin C and curcumin have been reported to be used in the treatment of different types of cancer, and successful results have been reported [169–174].

Studies have shown that many phytochemicals show more cytotoxic effects in various cancer cells than in normal cells [175–177]. Some studies have shown that polyphenols such as EGCG and genistein kill cancer cells more at the same dose with apoptosis than normal cells [178, 179]. We also showed that an herbal medicine named ankaferd, derived from different plant extracts, killed cancer cells more than normal cells at the same doses [180]. However, it is not clear how this differential effect occurs. This may be due to the difference in metabolism between cancer cells and normal cells. Since the metabolism of cancer cells is higher than normal cells, endogenous ROS production levels are much higher than normal cells [181]. The use of prooxidant phytochemicals emerges as an exciting strategy to target tumor cells selectively, due to further increase in ROS levels in cancer cells. Indeed, our findings show that the production of ROS in cancer cells to which phytochemicals are applied is significantly higher than normal cells and that there is a positive correlation between ROS level and cell death [163, 180]. The advantage of such a strategy is that it is not significantly affected by the fact that basal ROS levels of normal cells are lower than cancer cells and therefore less dependent on antioxidants. Although many *in vitro* cell culture studies have been carried out on the use of phytochemicals in the treatment of cancer, the number of experimental animals and clinical trials *in vivo* is low. An important problem in *in vivo* studies is that phytochemicals are digestion, absorption and bioavailability. Bioavailability is very low due to low absorption rate of many phytochemicals [182]. Therefore, enteral administration is preferred for cancer treatment [168, 171].

In conclusion, extensive researches over the past half a century have indicated that oxygen ROS play an important role in cancer metabolism. ROS are one of the main components of cell signaling pathways and have been shown to take roles in regulating cell transformation, survival, proliferation, invasion, angiogenesis, and metastasis [73, 168]. On the other hand, ROS can also suppress tumor progression. However, most chemotherapeutic and radiotherapeutic agents are designated to reduce the impact of ROS by augmenting ROS stress in cancer cells [183]. Due to these dual roles of ROS, both prooxidant-based and antioxidant-based anticancer agents have been developed [184]. It is clear that numerous chemotherapeutics mediate their effects by inducing ROS generation. However, unwarranted side effects of synthetic anticancer drugs should be minimized in healthy cells. For this reason, researchers continue to look at the nature and explore the potential for cancer treatment. A great number of phytochemicals including some of the vitamins, flavonoids, terpenoids, carotenoids, phenolics, phytoestrogens, minerals and antioxidants in plant materials are used for chemoprevention of cancer. However, various *in vitro* and *in vivo* experiments have shown that phytochemicals have carcinogenic potential as well as protective and curative effects against cancer. Many studies have shown that these three different effects of phytochemicals on cancer are related to the molecular structure of phytochemicals, bioavailability, dose and the oxidative status of the administered organism. For this reason, the molecular structure, bioavailability and

**5. Conclusion**

32 Reactive Oxygen Species (ROS) in Living Cells

Abdurrahim Kocyigit1 \*, Eray Metin Guler1 and Murat Dikilitas2

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Medical Biochemistry, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey

2 Department of Plant Protection, Faculty of Agriculture, Harran University, Sanliurfa, Turkey

#### **References**

[10] Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods: Impact on human health. Pharmacognosy Reviews. 2010;**4**(8):118

[25] Liu F-S. Mechanisms of chemotherapeutic drug resistance in cancer therapy—A quick review. Taiwanese Journal of Obstetrics and Gynecology. 2009;**48**(3):239-244

Role of Antioxidant Phytochemicals in Prevention, Formation and Treatment of Cancer

http://dx.doi.org/10.5772/intechopen.72217

35

[26] Howes RM. Dangers of antioxidants in cancer patients: A review. 2009. Available from:

[27] Ma H, Das T, Pereira S, Yang Z, Zhao M, Mukerji P, et al. Efficacy of dietary antioxidants combined with a chemotherapeutic agent on human colon cancer progression in a fluo-

[28] Payne AS, James WD,Weiss RB. Dermatologic toxicity of chemotherapeutic agents.

[29] Rybak LP, Mukherjea D, Jajoo S, Ramkumar V. Cisplatin ototoxicity and protection: Clinical and experimental studies. The Tohoku Journal of Experimental Medicine.

[30] Sak K. Chemotherapy and dietary phytochemical agents. Chemotherapy Research and

[31] Block G, Patterson B, Subar A. Fruit, vegetables, and cancer prevention: A review of the

[32] Zubair H, Azim S, Khan HY, Ullah MF, Wu D, Singh AP, et al. Mobilization of intracellular copper by gossypol and apogossypolone leads to reactive oxygen species-mediated cell death: Putative anticancer mechanism. International Journal of Molecular Sciences.

[33] Farhan M, Khan HY, Oves M, Al-Harrasi A, Rehmani N, Arif H, et al. Cancer therapy by catechins involves redox cycling of copper ions and generation of reactive oxygen spe-

[34] Temraz S, Mukherji D, Shamseddine A. Potential targets for colorectal cancer preven-

[35] Cieslak JA, Cullen JJ. Treatment of pancreatic cancer with pharmacological ascorbate.

[36] Fletcher A. Free radicals, antioxidants and eye diseases: Evidence from epidemiological studies on cataract and age-related macular degeneration. Ophthalmic Research.

[37] Poljšak B, Dahmane R. Free radicals and extrinsic skin aging. Dermatology Research and

[38] Luis A, Sandalio LM, Palma JM, Bueno P, Corpas FJ.Metabolism of oxygen radicals in peroxisomes and cellular implications. Free Radical Biology and Medicine. 2005;**39**(10):1289

[39] Inoue M, Sato EF, Nishikawa M, Park A-M, Kira Y, Imada I, et al. Mitochondrial generation of reactive oxygen species and its role in aerobic life. Current medicinal Chemistry.

tion. International Journal of Molecular Sciences. 2013;**14**(9):17279-17303

Current Pharmaceutical Biotechnology. 2015;**16**(9):759-770

epidemiological evidence. Nutrition and Cancer. 1992;**18**(1):1-29

rescent orthotopic mouse model. Anticancer Research. 2009;**29**(7):2421-2426

Seminars in Oncology, WB Saunders. 2006;**33**:86-97

Medi.Philica.com

2009;**219**(3):177-186

2016;**17**(6):973

Practice. 2012;**2012**:282570

cies. Toxins. 2016;**8**(2):37

2010;**44**(3):191-198

Practice. 2012;**2012**:135206

2003;**10**(23):2495-2505

[25] Liu F-S. Mechanisms of chemotherapeutic drug resistance in cancer therapy—A quick review. Taiwanese Journal of Obstetrics and Gynecology. 2009;**48**(3):239-244

[10] Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods:

[11] Lambert JD, Elias RJ. The antioxidant and pro-oxidant activities of green tea polyphenols: A role in cancer prevention. Archives of Biochemistry and Biophysics. 2010;**501**(1):65-72

[12] Ahmad A, Syed FA, Singh S, Hadi S. Prooxidant activity of resveratrol in the presence of copper ions: Mutagenicity in plasmid DNA. Toxicology Letters. 2005;**159**(1):1-12

[13] Khan HY, Zubair H, Ullah MF, Ahmad A, Hadi SM. Oral administration of copper to rats leads to increased lymphocyte cellular DNA degradation by dietary polyphenols:

Implications for a cancer preventive mechanism. Biometals. 2011;**24**(6):1169-1178 [14] Wätjen W, Michels G, Steffan B, Niering P, Chovolou Y, Kampkötter A, et al. Low concentrations of flavonoids are protective in rat H4IIE cells whereas high concentrations

cause DNA damage and apoptosis. The Journal of Nutrition. 2005;**135**(3):525-531

quercetin on A549 cells. Cell Biology International. 2007;**31**(10):1245-1250

and Medicine. 2004;**37**(3):287-303

2000;**408**(6809):239-247

34 Reactive Oxygen Species (ROS) in Living Cells

2009;**33**(6):431-434

[15] Robaszkiewicz A, Balcerczyk A, Bartosz G. Antioxidative and prooxidative effects of

[16] Galati G, O'brien PJ. Potential toxicity of flavonoids and other dietary phenolics: Significance for their chemopreventive and anticancer properties. Free Radical Biology

[17] Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature.

[18] Ames BN. Dietary carcinogens and anticarcinogens: Oxygen radicals and degenerative diseases. In: Risk Analysis in the Private Sector. USA: Springer; 1985. pp. 297-321 [19] Dufresne CJ, Farnworth ER. A review of latest research findings on the health promotion properties of tea. The Journal of Nutritional Biochemistry. 2001;**12**(7):404-421

[20] Islami F, Pourshams A, Nasrollahzadeh D, Kamangar F, Fahimi S, Shakeri R, et al. Tea drinking habits and oesophageal cancer in a high risk area in northern Iran: Population

[21] Ganesh B, Talole SD, Dikshit R. Tobacco, alcohol and tea drinking as risk factors for esophageal cancer: A case–control study from Mumbai, India. Cancer Epidemiology.

[22] Lambert JD, Kwon S-J, Hong J, Yang CS. Salivary hydrogen peroxide produced by holding or chewing green tea in the oral cavity. Free Radical Research. 2007;**41**(7):850-853

[23] Omenn GS, Goodman GE, Thornquist MD, Balmes J, Cullen MR, Glass A, et al. Risk factors for lung cancer and for intervention effects in CARET, the beta-carotene and retinol efficacy trial. JNCI: Journal of the National Cancer Institute. 1996;**88**(21):1550-1559 [24] Omenn GS, Goodman GE, Thornquist MD, Balmes J, Cullen MR, Glass A, et al. Risk factors for lung cancer and for intervention effects in CARET, the Beta-carotene and retinol efficacy trial. JNCI: Journal of the National Cancer Institute. 1996;**88**(21):1550-1559

based case-control study. British Medical Journal. 2009;**338**:b929

Impact on human health. Pharmacognosy Reviews. 2010;**4**(8):118

[40] Gogvadze V, Orrenius S, Zhivotovsky B. Mitochondria in cancer cells: What is so special about them? Trends in Cell Biology. 2008;**18**(4):165-173

[55] Liou G-Y, Storz P. Reactive oxygen species in cancer. Free Radical Research. 2010;

Role of Antioxidant Phytochemicals in Prevention, Formation and Treatment of Cancer

http://dx.doi.org/10.5772/intechopen.72217

37

[56] Prasad S, Gupta SC, Tyagi AK. Reactive oxygen species (ROS) and cancer: Role of anti-

[57] Acharya A, Das I, Chandhok D, Saha T. Redox regulation in cancer: A double-edged sword with therapeutic potential. Oxidative Medicine and Cellular Longevity. 2010;**3**(1):

[59] Wogan GN, Hecht SS, Felton JS, Conney AH, Loeb LA. Environmental and chemical carcinogenesis. Seminars in Cancer Biology, Academic Press. 2004;**14**(6):473-486

[60] Cooke MS, Evans MD, Dizdaroglu M, Lunec J. Oxidative DNA damage: Mechanisms,

[61] Dizdaroglu M, Kirkali G, Jaruga P. Formamidopyrimidines in DNA: Mechanisms of formation, repair, and biological effects. Free Radical Biology and Medicine. 2008;

[62] Kasai H. Analysis of a form of oxidative DNA damage, 8-hydroxy-2′-deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis. Mutation Research/Reviews

[63] Floyd RA, Watson JJ, Wong PK, Altmiller DH, Rickard RC. Hydroxyl free radical adduct of deoxyguanosine: Sensitive detection and mechanisms of formation. Free Radical

[64] Shibutani S, Takeshita M, Grollman AP. Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature. 1991;**349**(6308):431-434

[65] MQ D, Carmichael PL, Phillips DH. Induction of activating mutations in the human c-ha-ras-1 proto-oncogene by oxygen free radicals. Molecular Carcinogenesis. 1994;**11**(3):

[66] Dizdaroglu M, Jaruga P, Birincioglu M, Rodriguez H. Free radical-induced damage to DNA: Mechanisms and measurement 1, 2. Free Radical Biology and Medicine.

[67] Trachootham D, Lu W, Ogasawara MA, Valle NR-D, Huang P. Redox regulation of cell

[68] Suzuki K, Matsubara H. Recent advances in p53 research and cancer treatment. BioMed

[69] Sabharwal SS, Schumacker PT. Mitochondrial ROS in cancer: Initiators, amplifiers or an

survival. Antioxidants & Redox Signaling. 2008;**10**(8):1343-1374

Achilles' heel? Nature Reviews Cancer. 2014;**14**(11):709

oxidative nutraceuticals. Cancer Letters. 2017;**387**:95-105

[58] Oxyradicals LM. DNA damage. Carcinogenesis. 2000;**21**(3):361-370

mutation, and disease. The FASEB Journal. 2003;**17**(10):1195-1214

**44**(5):479-496

**45**(12):1610-1621

170-175

2002;**32**(11):1102-1115

in Mutation Research. 1997;**387**(3):147-163

Research Communications. 1986;**1**(3):163-172

Research International. 2011;**2011**:978312

23-34

[40] Gogvadze V, Orrenius S, Zhivotovsky B. Mitochondria in cancer cells: What is so special

[41] Storz P. Reactive oxygen species-mediated mitochondria-to-nucleus signaling: A key to

[42] Singh I. Mammalian peroxisomes: Metabolism of oxygen and reactive oxygen species.

[43] Mishina NM, Tyurin-Kuzmin PA, Markvicheva KN, Vorotnikov AV, Tkachuk VA, Laketa V, et al. Does Cellular Hydrogen Peroxide Diffuse or Act Locally?: Rochelle, NY,

[44] Szabó C, Ischiropoulos H, Radi R. Peroxynitrite: Biochemistry, pathophysiology and development of therapeutics. Nature Reviews Drug Discovery. 2007;**6**(8):662

[45] Bedard K, Krause K-H. The NOX family of ROS-generating NADPH oxidases: Phy-

[46] Chakravarthi S, Jessop CE, Bulleid NJ. The role of glutathione in disulphide bond formation and endoplasmic-reticulum-generated oxidative stress. EMBO Reports. 2006;

[47] Winterbourn CC. Reconciling the chemistry and biology of reactive oxygen species.

[48] Copin J-C, Gasche Y, Chan PH. Overexpression of copper/zinc superoxide dismutase does not prevent neonatal lethality in mutant mice that lack manganese superoxide dis-

[49] Bendayan M, Reddy J. Immunocytochemical localization of catalase and heat-labile enoyl-CoA hydratase in the livers of normal and peroxisome proliferator-treated rats. Laboratory Investigation; A Journal of Technical Methods and Pathology. 1982;

[50] Rhee SG, Kang SW, Netto LE, Seo MS, Stadtman ER. A family of novel peroxidases, per-

[51] Abeysinghe D, Li X, Sun C, Zhang W, Zhou C, Chen K. Bioactive compounds and antioxidant capacities in different edible tissues of citrus fruit of four species. Food Chemistry.

[52] Brigelius-Flohé R. Tissue-specific functions of individual glutathione peroxidases. Free

[53] Carlberg I, Mannervik B. Purification and characterization of the flavoenzyme glutathione reductase from rat liver. Journal of Biological Chemistry. 1975;**250**(14):5475-5480

[54] Townsend DM, Tew KD. The role of glutathione-S-transferase in anti-cancer drug resis-

siology and pathophysiology. Physiological Reviews. 2007;**87**(1):245-313

mutase. Free Radical Biology and Medicine. 2000;**28**(10):1571-1576

aging and radical-caused diseases. Science's STKE. 2006;**2006**(332):re3-re

Annals of the New York Academy of Sciences. 1996;**804**(1):612-627

about them? Trends in Cell Biology. 2008;**18**(4):165-173

USA: Mary Ann Liebert, Inc.; 2011

36 Reactive Oxygen Species (ROS) in Living Cells

Nature Chemical Biology. 2008;**4**(5):278-286

oxiredoxins. BioFactors. 1999;**10**(2-3):207-209

Radical Biology and Medicine. 1999;**27**(9):951-965

**7**(3):271-275

**47**(4):364-369

2007;**104**(4):1338-1344

tance. Oncogene. 2003;**22**(47):7369

[70] Su Z-Y, Shu L, Khor TO, Lee JH, Fuentes F, Kong A-NT.  In: Natural Products in Cancer Prevention and Therapy. Berlin Heidelberg: Springer; 2012. pp. 133-162

[86] Cheynier V, Comte G, Davies KM, Lattanzio V, Martens S. Plant phenolics: Recent advances on their biosynthesis, genetics, and ecophysiology. Plant Physiology and

Role of Antioxidant Phytochemicals in Prevention, Formation and Treatment of Cancer

http://dx.doi.org/10.5772/intechopen.72217

39

[87] de Pascual-Teresa S, Moreno DA, García-Viguera C. Flavanols and anthocyanins in cardiovascular health: A review of current evidence. International Journal of Molecular

[88] Tlili N, Mejri H, Yahia Y, Saadaoui E, Rejeb S, Khaldi A, et al. Phytochemicals and antioxidant activities of *Rhus tripartitum* (Ucria) fruits depending on locality and different

[89] Vinson JA, Su X, Zubik L, Bose P. Phenol antioxidant quantity and quality in foods:

[90] Steinmetz KA, Potter JD. Vegetables, fruit, and cancer. II. Mechanisms. Cancer Causes

[91] Scartezzini P, Antognoni F, Raggi M, Poli F, Sabbioni C. Vitamin C content and antioxidant activity of the fruit and of the ayurvedic preparation of Emblica officinalis Gaertn.

[92] Krinsky NI. Actions of carotenoids in biological systems. Annual Review of Nutrition.

[93] Harborne JB. Phenolic compounds. In: Phytochemical methods. Netherlands: Springer;

[94] Kühnau J. The flavonoids. A class of semi-essential food components: Their role in human nutrition. World Review of Nutrition and Dietetics. Karger Publishers. 1976;**24**:117-191

[95] Lila MA. Interactions between flavonoids that benefit human health. In: Anthocyanins.

[96] Yang CS, Landau JM, Huang M-T, Newmark HL. Inhibition of carcinogenesis by dietary

[97] Wang S, Meckling KA, Marcone MF, Kakuda Y, Tsao R. Can phytochemical antioxidant rich foods act as anti-cancer agents? Food Research International. 2011;**44**(9):2545-2554

[98] Bouayed J. Polyphenols: A potential new strategy for the prevention and treatment of

[99] Pandey KB, Rizvi SI. Plant polyphenols as dietary antioxidants in human health and

[100] Tan AC, Konczak I, Sze DM-Y, Ramzan I. Molecular pathways for cancer chemoprevention by dietary phytochemicals. Nutrition and Cancer. 2011;**63**(4):495-505

[101] Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. The international Journal

polyphenolic compounds. Annual Review of Nutrition. 2001;**21**(1):381-406

anxiety and depression. Current Nutrition & Food Science. 2010;**6**(1):13-18

disease. Oxidative Medicine and Cellular longevity. 2009;**2**(5):270-278

of Biochemistry & Cell Biology. 2007;**39**(1):44-84

fruits. Journal of Agricultural and Food Chemistry. 2001;**49**(11):5315-5321

Biochemistry. 2013;**72**:1-20

Sciences. 2010;**11**(4):1679-1703

& Control. 1991;**2**(6):427-442

1993;**13**(1):561-587

1984. pp. 37-99

stages of maturity. Food Chemistry. 2014;**160**:98-103

Journal of Ethnopharmacology. 2006;**104**(1):113-118

New York: Springer; 2008. pp. 306-323

[86] Cheynier V, Comte G, Davies KM, Lattanzio V, Martens S. Plant phenolics: Recent advances on their biosynthesis, genetics, and ecophysiology. Plant Physiology and Biochemistry. 2013;**72**:1-20

[70] Su Z-Y, Shu L, Khor TO, Lee JH, Fuentes F, Kong A-NT.  In: Natural Products in Cancer Prevention and Therapy. Berlin Heidelberg: Springer; 2012. pp. 133-162

[71] Trueba GP, Sánchez GM, Giuliani A. Oxygen free radical and antioxidant defense mech-

[72] Baldwin AS. Control of oncogenesis and cancer therapy resistance by the transcription

[73] Gupta SC, Hevia D, Patchva S, Park B, Koh W, Aggarwal BB. Upsides and downsides of reactive oxygen species for cancer: The roles of reactive oxygen species in tumorigenesis,

prevention, and therapy. Antioxidants & Redox Signaling. 2012;**16**(11):1295-1322

[74] Watson J. Oxidants, antioxidants and the current incurability of metastatic cancers.

[75] Schumacker PT.  Reactive oxygen species: A breath of life or death? Clinical

[77] Cabello CM, Bair 3rd W, Wondrak GT. Experimental therapeutics: Targeting the redox Achilles heel of cancer. Current Opinion in Investigational Drugs (London, England:

[78] Trachootham D, Zhou Y, Zhang H, Demizu Y, Chen Z, Pelicano H, et al. Selective killing of oncogenically transformed cells through a ROS-mediated mechanism by

[79] Raj L, Ide T, Gurkar AU, Foley M, Schenone M, Li X, et al. Selective killing of cancer cells with a small molecule targeting stress response to ROS. Nature. 2011;**475**(7355):231 [80] Syng-ai C, Kumari AL, Khar A. Effect of curcumin on normal and tumor cells: Role of

[81] Guzman ML, Rossi RM, Neelakantan S, Li X, Corbett CA, Hassane DC, et al. An orally bioavailable parthenolide analog selectively eradicates acute myelogenous leukemia

[82] Ameh SJ, Obodozie OO, Inyang US, Abubakar MS, Garba M. Current phytotherapy – A perspective on the science and regulation of herbal medicine. Journal of Medicinal

[83] Oz AT, Kafkas E. Phytochemicals in fruits and vegetables. In: Super Food and Functional Food - An Overview of Their Processing and Utilization. InTech; 2017. pp. 175-184 [84] Harborne JB. Classes and functions of secondary products from plants. Chemicals From

[85] Pereira DM, Valentão P, Pereira JA, Andrade PB. Phenolics: From chemistry to biology.

Molecular Diversity Preservation International. 2009;**14**(6):2202-2211

glutathione and bcl-2. Molecular Cancer Therapeutics. 2004;**3**(9):1101-1108

anism in cancer. Frontiers in Bioscience. 2004;**9**(10):2029-2044

factor NF-κB. Journal of Clinical Investigation. 2001;**107**(3):241

β-phenylethyl isothiocyanate. Cancer Cell. 2006;**10**(3):241-252

stem and progenitor cells. Blood. 2007;**110**(13):4427-4435

Open Biology. 2013;**3**(1):120144

38 Reactive Oxygen Species (ROS) in Living Cells

sword. Cancer Cell. 2006;**10**(3):175-176

Cancer Research. 2007;**13**(3):789-794

Plants Research. 2010;**4**(2):072-081

Plants. 1999. pp. 1-25

2000). 2007;**8**(12):1022-1037

[102] Azam S, Hadi N, Khan NU, Hadi SM. Prooxidant property of green tea polyphenols epicatechin and epigallocatechin-3-gallate: Implications for anticancer properties. Toxicology in Vitro. 2004;**18**(5):555-561

and differentiated HL-60 leukemia cells: Possible involvement of a redox mechanism.

Role of Antioxidant Phytochemicals in Prevention, Formation and Treatment of Cancer

http://dx.doi.org/10.5772/intechopen.72217

41

[116] Yeh S-L, Wang H-M, Chen P-Y, Wu T-C. Interactions of β-carotene and flavonoids on the secretion of pro-inflammatory mediators in an in vitro system. Chemico-Biological

[117] Liu RH. Potential synergy of phytochemicals in cancer prevention: Mechanism of

[118] Serafini M. The role of antioxidants in disease prevention. Medicine. 2006;**34**(12):533-535 [119] Bors W, Heller W, Michel C, Saran M. Flavonoids as antioxidants: Determination of radical-scavenging efficiencies. Methods in Enzymology. 1990;**186**:343-355

[120] Kocyigit A, Koyuncu I, Taskin A, Dikilitas M, Bahadori F, Turkkan B. Antigenotoxic and antioxidant potentials of newly derivatized compound naringenin-oxime relative to naringenin on human mononuclear cells. Drug and Chemical Toxicology. 2016;

[121] Sudheesh S, Sandhya C, Sarah Koshy A, Vijayalakshmi N. Antioxidant activity of flavonoids from *Solanum melongena*. Phytotherapy Research. 1999;**13**(5):393-396

[122] Van Acker SA, Tromp MN, Griffioen DH, Van Bennekom WP, Van Der Vijgh WJ, Bast A. Structural aspects of antioxidant activity of flavonoids. Free Radical Biology and

[123] Bouayed J, Bohn T. Exogenous antioxidants—Double-edged swords in cellular redox state: Health beneficial effects at physiologic doses versus deleterious effects at high

[124] Yordi EG, Pérez EM, Joao MM, Uriarte VE. Structural alerts for predicting clastogenic activity of pro-oxidant flavonoid compounds: quantitative structure-activity relation-

[125] Maurya DK, Devasagayam TPA. Antioxidant and prooxidant nature of hydroxycinnamic acid derivatives ferulic and caffeic acids. Food and Chemical Toxicology.

[126] Choi EJ, Chee K-M, Lee BH. Anti-and prooxidant effects of chronic quercetin adminis-

[127] Sahebkar A. Dual effect of curcumin in preventing atherosclerosis: The potential role of pro-oxidant – Antioxidant mechanisms. Natural Product Research. 2015;**29**(6):491-492

[128] Galati G, Sabzevari O, Wilson JX, O'Brien PJ. Prooxidant activity and cellular effects of the phenoxyl radicals of dietary flavonoids and other polyphenolics. Toxicology.

[129] Sharma M, Manoharlal R, Puri N, Prasad R. Antifungal curcumin induces reactive oxygen species and triggers an early apoptosis but prevents hyphae development by targeting the global repressor TUP1 in *Candida albicans*. Bioscience Reports.

tration in rats. European Journal of Pharmacology. 2003;**482**(1):281-285

doses. Oxidative Medicine and Cellular Longevity. 2010;**3**(4):228-237

ship study. Journal of Biomolecular Screening. 2012;**17**(2):216-224

International Journal of Cancer. 2002;**97**(5):593-600

action. The Journal of Nutrition. 2004;**134**(12):3479S-3485S

Interactions. 2009;**179**(2):386-393

Medicine. 1996;**20**(3):331-342

2010;**48**(12):3369-3373

2002;**177**(1):91-104

2010;**30**(6):391-404

**39**(1):66-73

and differentiated HL-60 leukemia cells: Possible involvement of a redox mechanism. International Journal of Cancer. 2002;**97**(5):593-600

[116] Yeh S-L, Wang H-M, Chen P-Y, Wu T-C. Interactions of β-carotene and flavonoids on the secretion of pro-inflammatory mediators in an in vitro system. Chemico-Biological Interactions. 2009;**179**(2):386-393

[102] Azam S, Hadi N, Khan NU, Hadi SM. Prooxidant property of green tea polyphenols epicatechin and epigallocatechin-3-gallate: Implications for anticancer properties. Toxi-

[103] Raza H, John A. Green tea polyphenol epigallocatechin-3-gallate differentially modulates oxidative stress in PC12 cell compartments. Toxicology and Applied Pharmacology.

[104] De Marchi U, Biasutto L, Garbisa S, Toninello A, Zoratti M. Quercetin can act either as an inhibitor or an inducer of the mitochondrial permeability transition pore: A demonstration of the ambivalent redox character of polyphenols. Biochimica et Biophysica

[105] Galati G, Lin A, Sultan AM, O'brien PJ. Cellular and in vivo hepatotoxicity caused by green tea phenolic acids and catechins. Free Radical Biology and Medicine. 2006;

[106] Metodiewa D, Jaiswal AK, Cenas N, Dickancaité E, Segura-Aguilar J. Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal

[107] Ochiai M, Nagao M, Wakabyashi K, Sugimura T. Superoxide dismutase acts as an enhancing factor for quercetin mutagenesis in rat-liver cytosol by preventing its decomposition. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis.

[108] Cao G, Sofic E, Prior RL. Antioxidant and prooxidant behavior of flavonoids: Structureactivity relationships. Free Radical Biology and Medicine. 1997;**22**(5):749-760

[109] Heim KE, Tagliaferro AR, Bobilya DJ. Flavonoid antioxidants: Chemistry, metabolism and structure-activity relationships. The Journal of Nutritional Biochemistry.

[110] Hanasaki Y, Ogawa S, Fukui S. The correlation between active oxygens scavenging and antioxidative effects of flavonoids. Free Radical Biology and Medicine. 1994;

[111] Gordon MH. The mechanism of antioxidant action in vitro. In: Food Antioxidants.

[112] Matés JM, Francisca M. Sánchez J. Role of reactive oxygen species in apoptosis: Implications for cancer therapy. The International Journal of Biochemistry & Cell

[113] Rice-Evans CA, Miller NJ, Paganga G. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine. 1996;**20**(7):933-956

[114] Rahman I, Biswas SK, Kirkham PA. Regulation of inflammation and redox signaling by

[115] Palozza P, Serini S, Torsello A, Boninsegna A, Covacci V, Maggiano N, et al. Regulation of cell cycle progression and apoptosis by β-carotene in undifferentiated

dietary polyphenols. Biochemical Pharmacology. 2006;**72**(11):1439-1452

product. Free Radical Biology and Medicine. 1999;**26**(1):107-116

cology in Vitro. 2004;**18**(5):555-561

Acta (BBA) – Bioenergetics. 2009;**1787**(12):1425-1432

2005;**207**(3):212-220

40 Reactive Oxygen Species (ROS) in Living Cells

**40**(4):570-580

1984;**129**(1):19-24

2002;**13**(10):572-584

Netherlands: Springer; 1990. pp. 1-18

Biology. 2000;**32**(2):157-170

**16**(6):845-850

[130] Puglia CD, Powell SR. Inhibition of cellular antioxidants: A possible mechanism of toxic cell injury. Environmental Health Perspectives. 1984;**57**:307

[145] Denning TL, Takaishi H, Crowe SE, Boldogh I, Jevnikar A, Ernst PB. Oxidative stress induces the expression of Fas and Fas ligand and apoptosis in murine intestinal epithe-

Role of Antioxidant Phytochemicals in Prevention, Formation and Treatment of Cancer

http://dx.doi.org/10.5772/intechopen.72217

43

[146] Uchikura K, Wada T, Hoshino S, Nagakawa Y, Aiko T, Bulkley GB, et al. Lipopolysaccharides induced increases in Fas ligand expression by Kupffer cells via mechanisms dependent on reactive oxygen species. American Journal of Physiology-

[147] Hampton MB, Orrenius S. Dual regulation of caspase activity by hydrogen peroxide:

[148] Gibson SB. A matter of balance between life and death: Targeting reactive oxygen species (ROS)-induced autophagy for cancer therapy. Autophagy. 2010;**6**(7):835-837 [149] Kong Q, Beel J, Lillehei K. A threshold concept for cancer therapy. Medical Hypotheses.

[150] Schafer FQ, Buettner GR. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radical Biology and

[151] Willett WC, Stampfer MJ. Current evidence on healthy eating. Annual Review of Public

[152] Shen L, Ji H-F. Theoretical study on physicochemical properties of curcumin. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2007;

[153] Aziz MH, Kumar R, Ahmad N. Cancer chemoprevention by resveratrol: In vitro and in vivo studies and the underlying mechanisms. International Journal of Oncology.

[154] Yordi EG, Pérez EM, Matos MJ, Villares EU. Antioxidant and pro-oxidant effects of polyphenolic compounds and structure-activity relationship evidence. In: Nutrition,

[155] Hadi S, Ullah M, Azmi A, Ahmad A, Shamim U, Zubair H, et al. Resveratrol mobilizes endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: A putative mechanism for chemoprevention of cancer. Pharmaceutical Research.

[156] Alexandre J, Batteux F, Nicco C, Chéreau C, Laurent A, Guillevin L, et al. Accumulation of hydrogen peroxide is an early and crucial step for paclitaxel-induced cancer cell death both in vitro and in vivo. International Journal of Cancer. 2006;**119**(1):41-48 [157] Alexandre J, Hu Y, Lu W, Pelicano H, Huang P. Novel action of paclitaxel against cancer cells: Bystander effect mediated by reactive oxygen species. Cancer Research.

[158] López-Lázaro M. A new view of carcinogenesis and an alternative approach to cancer

lial cells. Free Radical Biology and Medicine. 2002;**33**(12):1641-1650

Gastrointestinal and Liver Physiology. 2004;**287**(3):G620-G6G6

Implications for apoptosis. FEBS Letters. 1997;**414**(3):552-556

2000;**55**(1):29-35

Health. 2013;**34**:77-95

**67**(3):619-623

2003;**23**(1):17-28

2010;**27**(6):979-988

2007;**67**(8):3512-3517

Medicine. 2001;**30**(11):1191-1212

Wellbeing and Health. InTech; 2012. pp. 24-48

therapy. Molecular Medicine. 2010;**16**(3-4):144

[145] Denning TL, Takaishi H, Crowe SE, Boldogh I, Jevnikar A, Ernst PB. Oxidative stress induces the expression of Fas and Fas ligand and apoptosis in murine intestinal epithelial cells. Free Radical Biology and Medicine. 2002;**33**(12):1641-1650

[130] Puglia CD, Powell SR. Inhibition of cellular antioxidants: A possible mechanism of

[131] Halliwell B, Whiteman M. Measuring reactive species and oxidative damage in vivo and in cell culture: How should you do it and what do the results mean? British Journal

[132] Aruoma OI. Methodological considerations for characterizing potential antioxidant actions of bioactive components in plant foods. Mutation Research/Fundamental and

[133] Fukuhara K, Miyata N. Resveratrol as a new type of DNA-cleaving agent. Bioorganic &

[134] Zhou H, Zou P, Chen Z-C, You Y. A novel vicious cycle cascade in tumor chemother-

[135] Constantinou C, Papas A, Constantinou AI. Vitamin E and cancer: An insight into the anticancer activities of vitamin E isomers and analogs. International Journal of Cancer.

[136] Storz P. Reactive oxygen species in tumor progression. Frontiers in Bioscience. 2005;

[137] Block KI, Koch AC, Mead MN, Tothy PK, Newman RA, Gyllenhaal C. Impact of antioxidant supplementation on chemotherapeutic toxicity: A systematic review of the evidence from randomized controlled trials. International Journal of Cancer. 2008;

[138] Shukla A, Gulumian M, Hei TK, Kamp D, Rahman Q, Mossman BT. Multiple roles of oxidants in the pathogenesis of asbestos-induced diseases. Free Radical Biology and

[139] Renschler MF. The emerging role of reactive oxygen species in cancer therapy. European

[140] Berneis K, Bollag W, Kofler M, Lüthy H. The enhancement of the after effect of ionizing radiation by a cytotoxic methylhydrazine derivative. European Journal of Cancer.

[141] Azzam E, De Toledo S, Little J. Stress signaling from irradiated to non-irradiated cells.

[142] Simon H-U, Haj-Yehia A, Levi-Schaffer F. Role of reactive oxygen species (ROS) in

[143] Steller H. Mechanisms and genes of cellular suicide. Science-AAAS-Weekly Paper

[144] Wochna A, Niemczyk E, Kurono C, Masaoka M, Kędzior J, Słomińska E, et al. A possible role of oxidative stress in the switch mechanism of the cell death mode from apop-

tosis to necrosis-studies on ρ0 cells. Mitochondrion. 2007;**7**(1):119-124

toxic cell injury. Environmental Health Perspectives. 1984;**57**:307

Molecular Mechanisms of Mutagenesis. 2003;**523**:9-20

Medicinal Chemistry Letters. 1998;**8**(22):3187-3192

apy. Medical Hypotheses. 2007;**69**(6):1230-1233

2008;**123**(4):739-752

42 Reactive Oxygen Species (ROS) in Living Cells

**10**(1-3):1881-1896

**123**(6):1227-1239

Medicine. 2003;**34**(9):1117-1129

2004;**40**(13):1928-1933

Journal of Cancer. 2004;**40**(13):1934-1940

Current Cancer Drug Targets. 2004;**4**(1):53-64

Edition. 1995;**267**(5203):1445-1448

apoptosis induction. Apoptosis. 2000;**5**(5):415-418

of Pharmacology. 2004;**142**(2):231-255

[159] Smina T, Mohan A, Ayyappa K, Sethuraman S, Krishnan U. Hesperetin exerts apoptotic effect on A431 skin carcinoma cells by regulating mitogen activated protein kinases and cyclins. Cellular and Molecular Biology. 2015;**61**(6):92-99

[172] Ma Y, Chapman J, Levine M, Polireddy K, Drisko J, Chen Q. High-dose parenteral ascorbate enhanced chemosensitivity of ovarian cancer and reduced toxicity of chemo-

Role of Antioxidant Phytochemicals in Prevention, Formation and Treatment of Cancer

http://dx.doi.org/10.5772/intechopen.72217

45

[173] Hoffer LJ, Robitaille L, Zakarian R, Melnychuk D, Kavan P, Agulnik J, et al. Highdose intravenous vitamin C combined with cytotoxic chemotherapy in patients with

[174] Hosseini M, Hassanian SM, Mohammadzadeh E, ShahidSales S, Maftouh M, Fayazbakhsh H, et al. Therapeutic potential of curcumin in treatment of pancreatic cancer: Current status and future perspectives. Journal of Cellular Biochemistry. 2017;

[175] Ahmad N, Gupta S, Husain MM, Heiskanen KM, Mukhtar H. Differential antiproliferative and apoptotic response of sanguinarine for cancer cells versus normal cells.

[176] Babich H, Krupka M, Nissim HA, Zuckerbraun HL. Differential in vitro cytotoxicity of (−)-epicatechin gallate (ECG) to cancer and normal cells from the human oral cavity.

[177] Babich H, Pinsky S, Muskin E, Zuckerbraun H. In vitro cytotoxicity of a theaflavin mixture from black tea to malignant, immortalized, and normal cells from the human oral

[178] Ahmad N, Feyes DK, Agarwal R, Mukhtar H, Nieminen A-L. Green tea constituent epigallocatechin-3-gallate and induction of apoptosis and cell cycle arrest in human

carcinoma cells. Journal of the National Cancer Institute. 1997;**89**(24):1881-1886 [179] Chang K-L, Cheng H-L, Huang L-W, Hsieh B-S, Hu Y-C, Chih T-T, et al. Combined effects of terazosin and genistein on a metastatic, hormone-independent human pros-

[180] Kocyigit A, Guler EM, Haznedaroglu IC, Malkan UY. Ankaferd hemostat induces DNA damage, apoptosis and cytotoxic activity by generating reactive oxygen species in melanoma and normal cell lines. International Journal of Clinical and Experimental

[181] Agudo A, Cabrera L, Amiano P, Ardanaz E, Barricarte A, Berenguer T, et al. Fruit and vegetable intakes, dietary antioxidant nutrients, and total mortality in Spanish adults: Findings from the Spanish cohort of the European prospective investigation into cancer and nutrition (EPIC-Spain). The American Journal of Clinical Nutrition.

[182] Li Z, Jiang H, Xu C, Gu L. A review: Using nanoparticles to enhance absorption and bioavailability of phenolic phytochemicals. Food Hydrocolloids. 2015;**43**:153-164 [183] Tong L, Chuang C-C, Wu S, Zuo L. Reactive oxygen species in redox cancer therapy.

[184] Wang J, Yi J. Cancer cell killing via ROS: To increase or decrease, that is the question.

advanced cancer: A phase I-II clinical trial. PLoS One. 2015;**10**(4):e0120228

therapy. Science Translational Medicine. 2014;**6**(222):222ra18

Clinical Cancer Research. 2000;**6**(4):1524-1528

cavity. Toxicology in Vitro. 2006;**20**(5):677-688

tate cancer cell line. Cancer Letters. 2009;**276**(1):14-20

Toxicology in Vitro. 2005;**19**(2):231-242

Medicine. 2017;**10**(2):2116-2126

Cancer Letters. 2015;**367**(1):18-25

Cancer Biology & Therapy. 2008;**7**(12):1875-1884

2007;**85**(6):1634-1642

**118**(7):1634-1638

[172] Ma Y, Chapman J, Levine M, Polireddy K, Drisko J, Chen Q. High-dose parenteral ascorbate enhanced chemosensitivity of ovarian cancer and reduced toxicity of chemotherapy. Science Translational Medicine. 2014;**6**(222):222ra18

[159] Smina T, Mohan A, Ayyappa K, Sethuraman S, Krishnan U. Hesperetin exerts apoptotic effect on A431 skin carcinoma cells by regulating mitogen activated protein kinases and

[160] Zhao C, She T, Wang L, Su Y, Qu L, Gao Y, et al. Daucosterol inhibits cancer cell proliferation by inducing autophagy through reactive oxygen species-dependent manner.

[161] Ding W, Hu Z, Zhang Z, Ma Q, Tang H, Ma Z. Physapubescin B exhibits potent activity against human prostate cancer in vitro and in vivo. Journal of Agricultural and Food

[162] Gopalakrishnan A, Kong A-NT. Anticarcinogenesis by dietary phytochemicals: Cytoprotection by Nrf2 in normal cells and cytotoxicity by modulation of transcription factors NF-κB and AP-1 in abnormal cancer cells. Food and Chemical Toxicology.

[163] Kocyigit A, Koyuncu I, Dikilitas M, Bahadori F, Turkkan B. Cytotoxic, genotoxic and apoptotic effects of naringenin-oxime relative to naringenin on normal and cancer cell

[164] Kawai K, Tsuno NH, Kitayama J, Okaji Y, Yazawa K, Asakage M, et al. Epigallocatechin gallate induces apoptosis of monocytes. Journal of Allergy and Clinical Immunology.

[165] Larrosa M, Tomás-Barberán FA, Espín JC. The dietary hydrolysable tannin punicalagin releases ellagic acid that induces apoptosis in human colon adenocarcinoma Caco-2 cells by using the mitochondrial pathway. The Journal of Nutritional Biochemistry.

[166] Mertens-Talcott SU, Percival SS. Ellagic acid and quercetin interact synergistically with resveratrol in the induction of apoptosis and cause transient cell cycle arrest in human

[167] Kumar D, Basu S, Parija L, Rout D, Manna S, Dandapat J, et al. Curcumin and ellagic acid synergistically induce ROS generation, DNA damage, p53 accumulation and apoptosis in HeLa cervical carcinoma cells. Biomedicine & Pharmacotherapy. 2016;**81**:31-37

[168] Demiray M, Sahinbas H, Atahan S, Demiray H, Selcuk D, Yildirim I, et al. Successful treatment of c-kit-positive metastatic adenoid cystic carcinoma (ACC) with a combination of curcumin plus imatinib: A case report. Complementary Therapies in Medicine.

[169] A Cieslak J, J Cullen J. Treatment of pancreatic cancer with pharmacological ascorbate.

[170] Mata AMOF, Carvalho RM, Alencar MVOB, Cavalcante AACM, Silva BB. Ascorbic acid in the prevention and treatment of cancer. Revista da Associação Médica Brasileira.

[171] Stephenson CM, Levin RD, Spector T, Lis CG. Phase I clinical trial to evaluate the safety, tolerability, and pharmacokinetics of high-dose intravenous ascorbic acid in patients with advanced cancer. Cancer Chemotherapy and Pharmacology. 2013;**72**(1):139-146

leukemia cells. Cancer Letters. 2005;**218**(2):141-151

Current Pharmaceutical Biotechnology. 2015;**16**(9):759-770

lines. Asian Pacific Journal of Tropical Biomedicine. 2016;**6**(10):872-880

cyclins. Cellular and Molecular Biology. 2015;**61**(6):92-99

Life Sciences. 2015;**137**:37-43

44 Reactive Oxygen Species (ROS) in Living Cells

2008;**46**(4):1257-1270

2005;**115**(1):186-191

2006;**17**(9):611-625

2016;**27**:108-113

2016;**62**(7):680-686

Chemistry. 2015;**63**(43):9504-9512

**Section 2**

**Reactive Species Involvement in Pathology**

**Reactive Species Involvement in Pathology**

**Chapter 4**

**Provisional chapter**

**Reactive Oxygen Species and Bone Fragility**

**Reactive Oxygen Species and Bone Fragility**

DOI: 10.5772/intechopen.72305

Reactive oxygen species (ROS) are key signaling molecules that play an important role in the progression of inflammatory disorders. In the last decade, studies have indicated that ROS, including superoxide and hydrogen peroxide, are crucial components that regulate the differentiation process of osteoclasts. Osteoclasts (OCs), cells specialized for bone resorption, utilize ROS as second messengers during receptor activator of NF-κB ligand (RANKL)-induced differentiation and activation. The purpose of this chapter is to explore the current understanding of reactive oxygen species involvement in bone

**Keywords:** fragility fractures, free radicals, bone, osteoporosis, oxidative stress

Reactive oxygen species (ROS) are known to determine oxide-reducing balance alteration, oxidative stress, and carcinogenicity. Many diseases, including cancer and other pathologies associated, like arteriosclerosis and cataracts, are related to mitochondrial dysfunctions provoked

to the DNA and cell membranes, generating strong oxidant agents inside cells [3]. Reactive oxygen species (ROS) play a role in a number of degenerative conditions including osteoporosis [4]. Bone is an important organ performing three essential physiological functions: mechanical support, mineral homeostasis, and support of hematopoiesis. Bone diseases in the elderly are associated with increased morbidity and mortality. Osteoporosis is one of the most important diseases that affects the quality of life for the elderly [5]. Although the bones stop growing after adolescence, the bone is a very dynamic tissue. Bone tissue is continuously reabsorbed

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

O2

) react easily and cause damage

and reproduction in any medium, provided the original work is properly cited.

Nina Filip, Elena Cojocaru, Alexandru Filip,

Nina Filip, Elena Cojocaru, Alexandru Filip,

Additional information is available at the end of the chapter

by reactive oxygen species [1, 2]. Free radicals (O2−, HO•, H2

and regenerated by constantly changing its structure.

Additional information is available at the end of the chapter

Bogdan Veliceasa and Ovidiu Alexa

Bogdan Veliceasa and Ovidiu Alexa

http://dx.doi.org/10.5772/intechopen.72305

**Abstract**

pathophysiology.

**1. Introduction**

**Provisional chapter**

## **Reactive Oxygen Species and Bone Fragility**

**Reactive Oxygen Species and Bone Fragility**

DOI: 10.5772/intechopen.72305

Nina Filip, Elena Cojocaru, Alexandru Filip, Bogdan Veliceasa and Ovidiu Alexa Bogdan Veliceasa and Ovidiu Alexa Additional information is available at the end of the chapter

Nina Filip, Elena Cojocaru, Alexandru Filip,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72305

#### **Abstract**

Reactive oxygen species (ROS) are key signaling molecules that play an important role in the progression of inflammatory disorders. In the last decade, studies have indicated that ROS, including superoxide and hydrogen peroxide, are crucial components that regulate the differentiation process of osteoclasts. Osteoclasts (OCs), cells specialized for bone resorption, utilize ROS as second messengers during receptor activator of NF-κB ligand (RANKL)-induced differentiation and activation. The purpose of this chapter is to explore the current understanding of reactive oxygen species involvement in bone pathophysiology.

**Keywords:** fragility fractures, free radicals, bone, osteoporosis, oxidative stress

#### **1. Introduction**

Reactive oxygen species (ROS) are known to determine oxide-reducing balance alteration, oxidative stress, and carcinogenicity. Many diseases, including cancer and other pathologies associated, like arteriosclerosis and cataracts, are related to mitochondrial dysfunctions provoked by reactive oxygen species [1, 2]. Free radicals (O2−, HO•, H2 O2 ) react easily and cause damage to the DNA and cell membranes, generating strong oxidant agents inside cells [3]. Reactive oxygen species (ROS) play a role in a number of degenerative conditions including osteoporosis [4].

Bone is an important organ performing three essential physiological functions: mechanical support, mineral homeostasis, and support of hematopoiesis. Bone diseases in the elderly are associated with increased morbidity and mortality. Osteoporosis is one of the most important diseases that affects the quality of life for the elderly [5]. Although the bones stop growing after adolescence, the bone is a very dynamic tissue. Bone tissue is continuously reabsorbed and regenerated by constantly changing its structure.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

One of the most striking features of the bones is their ability to reshape. This remodeling process occurs during growth and continues throughout its life. During bone formation, the bone is formed and deposited, according to a particular pattern, by a process called ossification. The remodeling is continuous, leading to the organization of the bone structure in regular units, allowing the bone mass to gain maximum resistance to the mechanical forces acting on it. The older bone is removed by osteoclasts (bone-resorbing cells), and osteoblasts (bone-forming cells) lead to the deposition of new bone tissue. Osteoclasts secrete enzymes that break down the bone matrix as effectively as acids and convert calcium salts resulting in a soluble form (that can be absorbed into the bloodstream).

Osteoclast activity occurs behind the epiphyseal growth zone to reduce bone margins enlarged at the width of the bone stem that undergoes an elongation process.

The remodeling process is especially important for long bones supporting the limbs. These bones are widest at the ends and narrower in the middle, which gives added strength to the joints.

As osteoclasts destroy the old bone at the epithelial ends of the bone, the osteoblasts within the growth zone create a new epiphysis.

In each of the tubular spaces released by osteoclasts within the bone, the osteoblasts come in to deposit a new bone layer.

**2. Reactive oxygen species**

(figure from Ref. [5], reproduced with permission).

**2.1. ROS: general definitions**

), nitrogen oxide (NO), and iron (Fe2+) are present in erythrocytes. These elements

may in some conditions form reactive species, called radicals that affect red blood cells and vascular endothelium. A chemical species that presents an unpaired electron in outer orbitals and which can exist independently is called free radical. Radicals are very reactive trying to complete their own orbits by taking an electron from a neighboring molecule. Oxygen and nitrogen are the two chemical elements that in the body give rise to reactive species called the

**Figure 1.** Bone remodeling sequence executed by different bone cell types within basic multicellular units (BMUs)

Reactive species can be defined as atoms or molecules that contain in their structure an odd electron that causes an atom/molecule energy instability, which is reflected in their increased

Reactive species are produced in the body for physiological purposes in defense, vaso-relaxation, cellular or accidental exposure to toxic, or radiation exposure. When the amount of reactive species exceeds nontoxic physiological levels, the so-called oxidative stress occurs. Oxidative stress can be defined as the status of reactive oxygen or oxygen radicals in a biological system. It results from the imbalance between oxidants and antioxidants, in favor of

on the body called oxidative stress (OxS). The reactive oxygen species are constantly formed as reaction products in all cells by aerobic processes such as oxidative phosphorylation.

•−), hydrogen peroxide (H2

). Free oxygen radicals (ROS) produce harmful effects

O2

Reactive Oxygen Species and Bone Fragility http://dx.doi.org/10.5772/intechopen.72305 51

), hydroxyl

reactive oxygen species (ROS) and reactive nitrogen species (RNS), respectively.

oxidants, with destructive and pathogenic potential [7, 8].

O2

The reactive species of oxygen are superoxide (O2

radical (OH•), and singlet oxygen (1

Oxygen (O2

reactivity.

A bone that is poorly used, such as a lower immobilized member after a trauma, will be prone to reabsorption because bone destruction goes beyond bone formation.

Bones subjected to increased stress are permanently remodeled. The femur, for example, is completely replaced every 6 months.

Bone remodeling gives the different form of long bones. They are wider at each end and thinner in the middle. Bone remodeling not only changes the bone structure but also helps regulate the level of ionic calcium in the blood. Calcium is necessary for normal nervous transmission, for cell membrane formation, and for blood clotting. The resorption and bone formation sequence is shown in **Figure 1**.

Osteoblasts (bone-forming cells), osteoclasts (bone-resorbing cells), and osteocytes (loadsensing cells) are the cell types participating in bone remodeling. During remodeling, these cell types are spatially and temporally organized in functional structures called basic multicellular units (BMUs). The remodeling sequence operated by a BMU follows well-defined phases [5]. The bone remodeling sequence is influenced by a number of regulatory factors produced by hormonal glands, bone cells involved in sensing the mechanical environment, lymphocytes, and even tumor cells [6].

Osteoporosis is characterized by the parallel reduction of bone minerals and bone matrix so that the bone is in a low amount but with a normal percentage composition.

Despite rapid advances in our understanding over the last few years, the morbidity and mortality of patients resulting from this disease are still too high [4], and there is an urgent need for a proper assessment of the underlying mechanisms and the development of new treatment strategies to address this pathophysiological issue.

**Figure 1.** Bone remodeling sequence executed by different bone cell types within basic multicellular units (BMUs) (figure from Ref. [5], reproduced with permission).

#### **2. Reactive oxygen species**

One of the most striking features of the bones is their ability to reshape. This remodeling process occurs during growth and continues throughout its life. During bone formation, the bone is formed and deposited, according to a particular pattern, by a process called ossification. The remodeling is continuous, leading to the organization of the bone structure in regular units, allowing the bone mass to gain maximum resistance to the mechanical forces acting on it. The older bone is removed by osteoclasts (bone-resorbing cells), and osteoblasts (bone-forming cells) lead to the deposition of new bone tissue. Osteoclasts secrete enzymes that break down the bone matrix as effectively as acids and convert calcium salts resulting in

Osteoclast activity occurs behind the epiphyseal growth zone to reduce bone margins enlarged

The remodeling process is especially important for long bones supporting the limbs. These bones are widest at the ends and narrower in the middle, which gives added strength to the joints.

As osteoclasts destroy the old bone at the epithelial ends of the bone, the osteoblasts within

In each of the tubular spaces released by osteoclasts within the bone, the osteoblasts come in

A bone that is poorly used, such as a lower immobilized member after a trauma, will be prone

Bones subjected to increased stress are permanently remodeled. The femur, for example, is

Bone remodeling gives the different form of long bones. They are wider at each end and thinner in the middle. Bone remodeling not only changes the bone structure but also helps regulate the level of ionic calcium in the blood. Calcium is necessary for normal nervous transmission, for cell membrane formation, and for blood clotting. The resorption and bone

Osteoblasts (bone-forming cells), osteoclasts (bone-resorbing cells), and osteocytes (loadsensing cells) are the cell types participating in bone remodeling. During remodeling, these cell types are spatially and temporally organized in functional structures called basic multicellular units (BMUs). The remodeling sequence operated by a BMU follows well-defined phases [5]. The bone remodeling sequence is influenced by a number of regulatory factors produced by hormonal glands, bone cells involved in sensing the mechanical environment,

Osteoporosis is characterized by the parallel reduction of bone minerals and bone matrix so

Despite rapid advances in our understanding over the last few years, the morbidity and mortality of patients resulting from this disease are still too high [4], and there is an urgent need for a proper assessment of the underlying mechanisms and the development of new treat-

that the bone is in a low amount but with a normal percentage composition.

ment strategies to address this pathophysiological issue.

a soluble form (that can be absorbed into the bloodstream).

the growth zone create a new epiphysis.

completely replaced every 6 months.

formation sequence is shown in **Figure 1**.

lymphocytes, and even tumor cells [6].

to deposit a new bone layer.

50 Reactive Oxygen Species (ROS) in Living Cells

at the width of the bone stem that undergoes an elongation process.

to reabsorption because bone destruction goes beyond bone formation.

Oxygen (O2 ), nitrogen oxide (NO), and iron (Fe2+) are present in erythrocytes. These elements may in some conditions form reactive species, called radicals that affect red blood cells and vascular endothelium. A chemical species that presents an unpaired electron in outer orbitals and which can exist independently is called free radical. Radicals are very reactive trying to complete their own orbits by taking an electron from a neighboring molecule. Oxygen and nitrogen are the two chemical elements that in the body give rise to reactive species called the reactive oxygen species (ROS) and reactive nitrogen species (RNS), respectively.

#### **2.1. ROS: general definitions**

Reactive species can be defined as atoms or molecules that contain in their structure an odd electron that causes an atom/molecule energy instability, which is reflected in their increased reactivity.

Reactive species are produced in the body for physiological purposes in defense, vaso-relaxation, cellular or accidental exposure to toxic, or radiation exposure. When the amount of reactive species exceeds nontoxic physiological levels, the so-called oxidative stress occurs. Oxidative stress can be defined as the status of reactive oxygen or oxygen radicals in a biological system. It results from the imbalance between oxidants and antioxidants, in favor of oxidants, with destructive and pathogenic potential [7, 8].

The reactive species of oxygen are superoxide (O2 •−), hydrogen peroxide (H2 O2 ), hydroxyl radical (OH•), and singlet oxygen (1 O2 ). Free oxygen radicals (ROS) produce harmful effects on the body called oxidative stress (OxS). The reactive oxygen species are constantly formed as reaction products in all cells by aerobic processes such as oxidative phosphorylation.

The base state of oxygen is triplet. This means that it has two unpaired electrons that are positioned on two different molecular orbitals.

Because electrons have parallel spin, oxygen can only rotate in the same orbit. Since molecular oxygen is paramagnetic, it is concluded that it has the structure represented in the right-hand side of **Figure 2**. Molecular oxygen can react rapidly with single unpaired electrons of transitory metals (e.g., Fe, Cu, Mn). One mole of properly chelated copper could catalyze consumption of the oxygen in an average room within 1 s in [9]. Depending on the conditions, O2 can be both kinetically stable (not reactive) and very reactive.

For proteins and enzymes that have a metal in their structure, the oxidative attack of O2 is slow, and in conclusion the superoxide radical will slowly form.

The reaction involves two steps:

**Figure 4.** Reactive oxygen species generation.

tion reaction.

species. H2

the cytosol).

**Singlet oxygen** (1

**Hydrogen peroxide** (H2

O2

it can initiate chain reaction.

O2

rated fatty acids, cholesterol-forming hydroperoxides.

The nonenzymatic reaction occurring in living cells is:

O2

O2 + Fe3+ → O2 + Fe2+ (1)

Fe2+ + H2 O2 → Fe3+ + OH− + OH• (2)

O2 + H2 O2 → OH• + OH− + O2 (3)

Most of the superoxide radicals are formed by electrons derived from the coenzyme Q cycle in complex III and by NADH flavoprotein dehydrogenase (complex I). The superoxide radical, which has a relatively low degree of toxicity compared to other reactive oxygen species, is kept undetected by enzymes called superoxide dismutase (SOD) that catalyzes the dismuta-

in the structure, which gives it reactive stability, being the most easily measurable reactive

tions or oxidase-catalyzed reactions. This process takes place at the level of peroxisomes, cellular organisms with rich enzyme equipment. Concentration of hydrogen peroxide is controlled by catalase (present in peroxisomes) and by glutathione peroxidase (localized to

**Hydroxyl radical** (OH•) is a highly reactive free radical which diffuses over small distances until it reacts with any organic molecule encountered such as carbohydrates, amino acids, lipids, nucleic acids, and organic acids. The toxicity of this molecule comes from the fact that

than the hydroxyl radical by reacting with many organic compounds such as polyunsatu-

is a weak oxidizing agent that forms as a result of the O2

) is not a free radical due to the fact that it has no unpaired electron

) can result from oxidoreduction reactions. Single oxygen is more reactive

Reactive Oxygen Species and Bone Fragility http://dx.doi.org/10.5772/intechopen.72305 53

In the presence of ferric iron, superoxide radical is a source of hydroxyl radical.

Superoxide (O2 ): this radical constitutes the first step of oxygen activation by way of univalent reduction, forming in most of the autoxidation reactions by the capture of an electron that is placed on one of the antiknocking orbital n (**Figure 3**).

After an electron has been acquired, the following electrons are added more easily, and the reactions take place faster, leading to the generation of reactive oxygen species [10].

Reducing the hydroxyl radical to water takes place in the last stage of electron acceptance (**Figure 4**).

Superoxide radical (O2 ) can react in the presence of ferric iron with hydrogen peroxide generating the most potent hydroxyl radical through a nonenzymatic reaction named Haber-Weiss reaction.

**Figure 2.** The structure of oxygen molecule.

**Figure 3.** Superoxide radical generation.

**Figure 4.** Reactive oxygen species generation.

The base state of oxygen is triplet. This means that it has two unpaired electrons that are posi-

Because electrons have parallel spin, oxygen can only rotate in the same orbit. Since molecular oxygen is paramagnetic, it is concluded that it has the structure represented in the right-hand side of **Figure 2**. Molecular oxygen can react rapidly with single unpaired electrons of transitory metals (e.g., Fe, Cu, Mn). One mole of properly chelated copper could catalyze consumption of the oxygen in an average room within 1 s in [9]. Depending on the conditions, O2

For proteins and enzymes that have a metal in their structure, the oxidative attack of O2

reduction, forming in most of the autoxidation reactions by the capture of an electron that is

After an electron has been acquired, the following electrons are added more easily, and the

Reducing the hydroxyl radical to water takes place in the last stage of electron acceptance

ating the most potent hydroxyl radical through a nonenzymatic reaction named Haber-Weiss

reactions take place faster, leading to the generation of reactive oxygen species [10].

): this radical constitutes the first step of oxygen activation by way of univalent

) can react in the presence of ferric iron with hydrogen peroxide gener-

can

is

tioned on two different molecular orbitals.

52 Reactive Oxygen Species (ROS) in Living Cells

Superoxide (O2

(**Figure 4**).

reaction.

Superoxide radical (O2

**Figure 3.** Superoxide radical generation.

**Figure 2.** The structure of oxygen molecule.

be both kinetically stable (not reactive) and very reactive.

placed on one of the antiknocking orbital n (**Figure 3**).

slow, and in conclusion the superoxide radical will slowly form.

The reaction involves two steps:

$$\rm O\_2 + Fe^{3+} \rightarrow \rm O\_2 + Fe^{2+} \tag{1}$$

$$\text{Fe}^{2+} + \text{H}\_2\text{O}\_2 \rightarrow \text{Fe}^{3+} + \text{OH}^- + \text{OH}^\* \tag{2}$$

The nonenzymatic reaction occurring in living cells is:

$$\rm O\_2 + H\_2O\_2 \rightarrow OH^\* + OH^- + O\_2 \tag{3}$$

In the presence of ferric iron, superoxide radical is a source of hydroxyl radical.

Most of the superoxide radicals are formed by electrons derived from the coenzyme Q cycle in complex III and by NADH flavoprotein dehydrogenase (complex I). The superoxide radical, which has a relatively low degree of toxicity compared to other reactive oxygen species, is kept undetected by enzymes called superoxide dismutase (SOD) that catalyzes the dismutation reaction.

**Hydrogen peroxide** (H2 O2 ) is not a free radical due to the fact that it has no unpaired electron in the structure, which gives it reactive stability, being the most easily measurable reactive species. H2 O2 is a weak oxidizing agent that forms as a result of the O2 -dismutation reactions or oxidase-catalyzed reactions. This process takes place at the level of peroxisomes, cellular organisms with rich enzyme equipment. Concentration of hydrogen peroxide is controlled by catalase (present in peroxisomes) and by glutathione peroxidase (localized to the cytosol).

**Hydroxyl radical** (OH•) is a highly reactive free radical which diffuses over small distances until it reacts with any organic molecule encountered such as carbohydrates, amino acids, lipids, nucleic acids, and organic acids. The toxicity of this molecule comes from the fact that it can initiate chain reaction.

**Singlet oxygen** (1 O2 ) can result from oxidoreduction reactions. Single oxygen is more reactive than the hydroxyl radical by reacting with many organic compounds such as polyunsaturated fatty acids, cholesterol-forming hydroperoxides.

Singlet oxygen can be formed from superoxide radical in [11]:

$$2\text{O}\_2 + 2\text{H}^+ \to \text{H}\_2\text{O}\_2 + ^1\text{O}\_2\tag{4}$$

of lipids, the peroxyl radicals are formed by chain reactions. These intermediates that are formed can amplify the lesion. The attack of ROS affects both erythrocytes and endothelial cells. DNA damage may occur in nucleus containing endothelium cell besides lipid and protein oxidation. Interaction between free radicals and DNA can lead to strand breaks or

Reactive Oxygen Species and Bone Fragility http://dx.doi.org/10.5772/intechopen.72305 55

In general, lipid peroxidation occurs in late stages of aggression, so therapies directed against lipoperoxidation may be less beneficial. This is another reason that can lead to erroneous con-

As long as they do not accumulate in excess, ROS have positive effects in the body, being involved in various cellular mechanisms. For example, biosynthesis of thyroid hormones involves the formation of hydrogen peroxide in order to assemble thyroglobulin iodine to synthesize thyroxine. On the other hand, in the immune response, in neutrophils and macrophages, NADPH oxidase catalyzes the formation of superoxide anion by internalizing

structural changes such as adduct formation (**Figure 5**) [21, 22].

**Figure 5.** ROS damaging activity.

**Figure 6.** Cells response under ROS attack [23].

clusions about the importance of oxidative stress and antioxidant therapies.

Although it is not a radical, singlet oxygen is more reactive than the hydroxyl radical. The most reactive radical is hydroxyl (OH•) which takes electrons from other molecules around it, whereas superoxide (O2 ) and hydrogen peroxide (H2 O2 ) are more selective in their reactions with various molecules [12].

The processes so far presented above take place in all human cells. The molecular oxygen that is transported to the tissues is found for a short period of time unbound. It is assumed that in this state, it generates the radicals previously presented. Oxygen binds to hemoglobin molecule at the ferrous iron (Fe2+). Some of Fe2+ is transformed by O2 to ferric form (Fe3+) in resulting methemoglobin. Methemoglobin reductase is an enzymatic system which restores Fe3+ to Fe2+ and reduces methemoglobin back to hemoglobin. Oxygenated hem has some of the electronic characteristics of a Fe3+OO peroxide anion [10]. Like in this study [13], the author found that the Fe3+O2− complex is able to generate superoxide radical during the normal molecular oxygen transport to tissues through the hemoglobin auto-oxidation.

Some researchers propose that hemoglobin may have oxidative reaction in the oxygen-releasing process.

Like in this study from 2006 [14], the author demonstrates that at intermediate oxygen pressure, where hemoglobin partially releases molecular oxygen, the superoxide radical production increases. They show that superoxide radical is released in the hydrophobic hem pocket.

Through the Haber-Weiss reaction, H2 O2 , even if it is not a radical, can form short and very active hydroxyl radicals [15]. The hydroxyl radical (OH•), which is highly reactive, reacts with any biomolecules it encounters. There are studies according to which the high reactivity of the hydroxyl radical decreases its ability to diffuse [16]. Since the hydroxyl radical may generate an autocatalytic reaction in the chain, we can say that it is particularly dangerous.

There are some researchers who believe that the hydroxyl radical is responsible for cellular damage and others that ferric ion promotes initiation of the chain reaction [17–19]. In conclusion, both oxidative species can form in living cells [20].

Like in **Figure 4**, ROS are produced accidentally and physiologically in various enzyme-catalyzed reactions.

#### **2.2. Effects and signaling of ROS**

Reactive oxygen species can interact with any biological molecule causing damage to lipids, proteins, and DNA. Prospects for effective therapeutic intervention may fail if the initial target of oxidative stress is unknown. For example, it is known that DNA is the primary target of lesions produced by the addition of H2 O2 to mammalian cell cultures, so that DNA strand breaks occur before lipid peroxides or "detectable" oxidized proteins. The method of detecting lesions produced on target molecules may give incomplete information. The evidence of protein damage by detecting carbonyl radicals in the initial stages of lesions may be negative, but the determination of SH oxidation, which occurs earlier, is positive. During peroxidation

**Figure 5.** ROS damaging activity.

Singlet oxygen can be formed from superoxide radical in [11]:

cule at the ferrous iron (Fe2+). Some of Fe2+ is transformed by O2

gen transport to tissues through the hemoglobin auto-oxidation.

Although it is not a radical, singlet oxygen is more reactive than the hydroxyl radical. The most reactive radical is hydroxyl (OH•) which takes electrons from other molecules around it,

The processes so far presented above take place in all human cells. The molecular oxygen that is transported to the tissues is found for a short period of time unbound. It is assumed that in this state, it generates the radicals previously presented. Oxygen binds to hemoglobin mole-

methemoglobin. Methemoglobin reductase is an enzymatic system which restores Fe3+ to Fe2+ and reduces methemoglobin back to hemoglobin. Oxygenated hem has some of the electronic characteristics of a Fe3+OO peroxide anion [10]. Like in this study [13], the author found that the Fe3+O2− complex is able to generate superoxide radical during the normal molecular oxy-

Some researchers propose that hemoglobin may have oxidative reaction in the oxygen-releas-