**3. Bioflavonoids and other natural compounds as Top2 inhibitors**

A class of chemical compounds called bioflavonoids are contained in soy, fruits, vegetables, tea, coffee, wine, energy drinks, and dietary supplements [21–27]. Bioflavonoids are characterized by multiple phenolic rings that are central to their ability to inhibit the enzyme Top2 in a similar manner to the chemotherapeutic drug etoposide [16, 28, 29]. Some pesticides and flame retardants also contain multiple phenolic rings and have been identified as Top2 inhibitors. Bioflavonoids are separated into 12 different sub-classes based upon their structure; however only six are contained in dietary sources: flavanols, flavonols, flavones, isoflavones, flavanones, and anthocyanidins (**Figure 3**) [60, 61].

### **3.1 Isoflavones**

Isoflavones are polyphenolic secondary plant metabolites produced through the flavonoid-producing phenyl-propanoid synthesis pathway (**Figure 4**). In order for isoflavone production, the plant must express the isoflavone synthase enzyme which converts flavanone precursors into isoflavones. This isoflavone synthase is only expressed in legumes and a few other select species. Plants with the highest concentrations of isoflavones are soy, red clover, and kudzu. The amount of isoflavone depends upon the conditions the plants were grown, and the final concentration of isoflavones in food products (including dietary supplements) depends upon which portion of the plant is used and the processing methods. Genistein, daidzein, glycitein, formononetin, biochanin A and irilone are the main isoflavones isolated

#### **Figure 3.**

*Basic chemical structures of dietary bioflavonoids. The middle circled backbone represents the general bioflavonoid poly-phenol ring structure. The six structures surrounding show the general structural differences between the sub-groups.*

**81**

**3.2 Flavones**

[23, 64, 65].

**Figure 4.**

*and Myricetin, and flavones: Luteolin.*

who ingest only 0.1–3.3 mg/day [66, 67] .

progression and cell proliferation genes [62].

*DNA Damage and Repair Mechanisms Triggered by Exposure to Bioflavonoids and Natural…*

from plants [60, 62, 63]. Genistein and daidzein are of particular interest due to their high concentration in soy products [60]. Genistein is an estrogen derivative available at health food stores as dietary and menopausal supplements, and a soy phytoestrogen present in foods, particularly soybeans, and infant soy formulas

*Structure of commonly found bioflavonoids flavanols: Genistein and Daidzein, flavonols: Kaemferol quercetin* 

Interest in isoflavones has spiked in the past 20 years. This is due to the attribution of consumption of isoflavone-containing products with lower occurrences of coronary heart disease, breast and prostate cancer. This hypothesis derived from observations that citizens of Asian countries have lower incidence of these diseases compared to citizens of Western countries, and that citizens in Asian countries typically ingest 8-50 mg/day of isoflavones compared to citizens in Western countries

Due to this potential health relevance, studies examined the impact of high intake of isoflavones, but the results have been inconclusive [62]. In animal models, increased genistein intake resulted in increased rates of pituitary and mammary gland tumors and stimulated MCF-7 tumor growth. Additionally, while increased genistein intake in post-menopausal women in Asian countries decreased breast cancer risk, this decreased risk was not sustained in post-menopausal women in Western countries, including both native inhabitants and Asian immigrants. Some studies, particularly of British women, showed that increased serum genistein levels in women with early stage breast cancer had increased transcription of cell cycle

Flavones are the end product of a complex multi-step synthetic pathway that occurs within a wide variety of plants (**Figure 4**). This pathway begins with phenylalanine that is converted through the generalized phenylpropanoid pathway that synthesizes most flavonoids. Subsequently, p-coumaroyl-CoA must be synthesized into chalcone with chalcone synthase. Chalcone can be isomerized into a flavanone

*DOI: http://dx.doi.org/10.5772/intechopen.95453*

*DNA Damage and Repair Mechanisms Triggered by Exposure to Bioflavonoids and Natural… DOI: http://dx.doi.org/10.5772/intechopen.95453*

#### **Figure 4.**

*DNA - Damages and Repair Mechanisms*

and anthocyanidins (**Figure 3**) [60, 61].

**3.1 Isoflavones**

**3. Bioflavonoids and other natural compounds as Top2 inhibitors**

vegetables, tea, coffee, wine, energy drinks, and dietary supplements [21–27]. Bioflavonoids are characterized by multiple phenolic rings that are central to their ability to inhibit the enzyme Top2 in a similar manner to the chemotherapeutic drug etoposide [16, 28, 29]. Some pesticides and flame retardants also contain multiple phenolic rings and have been identified as Top2 inhibitors. Bioflavonoids are separated into 12 different sub-classes based upon their structure; however only six are contained in dietary sources: flavanols, flavonols, flavones, isoflavones, flavanones,

Isoflavones are polyphenolic secondary plant metabolites produced through the flavonoid-producing phenyl-propanoid synthesis pathway (**Figure 4**). In order for isoflavone production, the plant must express the isoflavone synthase enzyme which converts flavanone precursors into isoflavones. This isoflavone synthase is only expressed in legumes and a few other select species. Plants with the highest concentrations of isoflavones are soy, red clover, and kudzu. The amount of isoflavone depends upon the conditions the plants were grown, and the final concentration of isoflavones in food products (including dietary supplements) depends upon which portion of the plant is used and the processing methods. Genistein, daidzein, glycitein, formononetin, biochanin A and irilone are the main isoflavones isolated

*Basic chemical structures of dietary bioflavonoids. The middle circled backbone represents the general bioflavonoid poly-phenol ring structure. The six structures surrounding show the general structural differences* 

A class of chemical compounds called bioflavonoids are contained in soy, fruits,

**80**

**Figure 3.**

*between the sub-groups.*

*Structure of commonly found bioflavonoids flavanols: Genistein and Daidzein, flavonols: Kaemferol quercetin and Myricetin, and flavones: Luteolin.*

from plants [60, 62, 63]. Genistein and daidzein are of particular interest due to their high concentration in soy products [60]. Genistein is an estrogen derivative available at health food stores as dietary and menopausal supplements, and a soy phytoestrogen present in foods, particularly soybeans, and infant soy formulas [23, 64, 65].

Interest in isoflavones has spiked in the past 20 years. This is due to the attribution of consumption of isoflavone-containing products with lower occurrences of coronary heart disease, breast and prostate cancer. This hypothesis derived from observations that citizens of Asian countries have lower incidence of these diseases compared to citizens of Western countries, and that citizens in Asian countries typically ingest 8-50 mg/day of isoflavones compared to citizens in Western countries who ingest only 0.1–3.3 mg/day [66, 67] .

Due to this potential health relevance, studies examined the impact of high intake of isoflavones, but the results have been inconclusive [62]. In animal models, increased genistein intake resulted in increased rates of pituitary and mammary gland tumors and stimulated MCF-7 tumor growth. Additionally, while increased genistein intake in post-menopausal women in Asian countries decreased breast cancer risk, this decreased risk was not sustained in post-menopausal women in Western countries, including both native inhabitants and Asian immigrants. Some studies, particularly of British women, showed that increased serum genistein levels in women with early stage breast cancer had increased transcription of cell cycle progression and cell proliferation genes [62].

#### **3.2 Flavones**

Flavones are the end product of a complex multi-step synthetic pathway that occurs within a wide variety of plants (**Figure 4**). This pathway begins with phenylalanine that is converted through the generalized phenylpropanoid pathway that synthesizes most flavonoids. Subsequently, p-coumaroyl-CoA must be synthesized into chalcone with chalcone synthase. Chalcone can be isomerized into a flavanone

by chalcone isomerase. Finally, flavone synthase class I or II enzymes catalyze the synthesis of a flavone from flavanones. Flavones, similar to flavonols, can protect the plant from UV-B radiation. Flavones have the additional ability to provide protection against biological attacks from pathogenic microbes by acting as signaling molecules to activate differential gene transcription to prevent the growth of microorganisms after invasion. Additionally, flavones can be expressed to deter insects and nematodes from eating the plant or to interfere with the growth and reproduction of other plants [60].

Flavones are found across a variety of plant species, and expression of flavones appears to be widespread within the plant, from the roots to the leaves. However, though flavones are found throughout the plant kingdom, they are found much less commonly in fruits and vegetables as compared to flavonols. Apigenin and luteolin are the main flavonols contained in food sources including celery, parsley, thyme, red peppers, and fruit skins [61, 68]. In humans, flavones, much like isoflavones and flavonols, seem to have antioxidant and anti-tumor capabilities and to affect signal transduction pathways [69].

#### **3.3 Flavonols**

Flavonols are primarily in fruits, vegetables, red wine, and tea and they compose the largest portion of humans' bioflavonoid intake given their distribution across a wide number of plant species (**Figure 4**) [61]. Within plants it has been shown that flavonols have the ability to protect the plant against UV-B damage, and they protect the plants against oxidative damage with their antioxidant capability [70, 71]. Scientists and physicians want to determine ways to utilize the antioxidant capability of flavonols in human populations as a protectant against cardiovascular and neurological disease and against exercise induced oxidation in smokers and athletes [72, 73].

The most common flavonols in foods are quercetin, kaempferol, myricetin, and fiestin, with a majority of published literature focusing upon the first three. Similar to isoflavones the concentration of flavonol in the food product depends upon the plant, the growth conditions, and the part of the plant used. Flavonols are found in highest concentrations in the leaves, flowers, and fruits, which are exposed to sunlight; the exception to this being onions which grow below ground [70, 71]. The human dietary source of flavonols is dependent on culture and region. Humans residing in Asian countries typically ingest flavonols through green tea, while the Netherlands, United States and Denmark inhabitants mainly ingest them from onions, apples, and tea. Citizens of Mediterranean areas ingest flavonols from green vegetables. Within Italy, red wine is the main source of flavonols, though inhabitants of Northern villages also have a high intake from salads, soups, fruits. The prevalence of flavonols in the human diet has produced a large interest in understanding their multiple cellular effects and potential impact on human health [70].

#### **3.4 Additional compounds as Top2 inhibitors**

Additional natural compounds other than bioflavonoids may also act as inhibitors of Top2. Bakuchicin from the furanocoumarin family is present in fruits and legumes [74]. In research conducted to study DNA-polymerase inhibition activity of *Psoralea corylifolia L*. (Leguminosae), bakuchincin was found to be a weak Top2 inhibitor [75]. Additional reported naturally occurring Top1 and Top2 inhibitors are benzophenone compounds such as xanthochymol and Garcinol at effective concentrations comparable to those of etoposide (∼25 − 100 μM) [76, 77]. A comparative study between the naturally occurring constituent of black seed thymoquinone

**83**

*DNA Damage and Repair Mechanisms Triggered by Exposure to Bioflavonoids and Natural…*

used as a spice in eastern cooking and a known Top2 inhibitor 1,4-benzoquinone showed structural and functional similarity between the two compounds and the

Triterpenoids are present in plants, widely distributed within the root, stem, leaves, bark. They are components in the waxy covering of fruits and herbs such as jujube, lavender, and thyme [79]. Triterpenoids have two major components, C5 units and isopentyl diphosphate [80], and are generally present as saponins that act as defense chemicals for protection against microbes. Triterpenoids betulin lupane and oleanane from the bark of *Phyllanthus flexuosus*, derivatives of betulinc acid, and oxygenated derivatives of oleanane called celastroloids were reported to act as human Top2 inhibitors to varying degrees [81–84]. In addition, betulinic acid which is an oxidative derivative of betullin inhibits cell proliferation by inhibiting

Halogenated compounds in household and baby products include polychlorinated biphenyls (PCBs), detectable in indoor carpets, and polybrominated diphenyl ethers (PBDEs), used as flame retardants, increase DNA cleavage by TopIIα in vitro and in cultured human cells [85]. Recent CRISPR-Cas9 screening against a large panel of genotoxic agents identified the synthetic small molecule pyridostatin as a Top2 inhibitor. Pyridostatin is a G-quadruplex stabilizer and this stabilization

topoisomerase-DNA binding and suppressing NF-κB activation [83].

**4. Flavonols, flavonols, flavones, isoflavones, flavanones, and** 

individual bioflavonoids can act through one or both mechanisms [29, 86].

weak traditional poison, but kaempferol does not have this activity [29, 87].

Flavanols, flavonols, flavones, flavanones, and anthocyanidins (but not isoflavones) have the potential to act as strong covalent Top2 poisons [86]. A covalent Top2 poison works in a redox-dependent manner, binding to a distal site on the Top2 enzyme and increasing its ability to cause a DSB in step 2 of the catalytic multi-step reaction through conformational changes to the enzyme. The key structural component for a covalent poison is having 3 –OH groups on the B ring of the bioflavonoid structure. However, it is likely bioflavonoids with 2 –OH groups on the B ring act as a weak covalent poison and the ability to act as a covalent poison increases with more –OH groups (**Figure 5**) [49–50]. A 4'-OH group on the B ring is necessary for binding, and 3′ and 5'-OH groups improve covalent binding strength. Thus, a strong covalent poison contains 3 –OH groups on the B ring of the bioflavonoid structure. For example, among the flavonols, structure predicts that myricetin has high activity, quercetin has intermediate activity, and kaempferol has weak activity, if at all, as a covalent Top2 poison (**Figure 4**). Cell free studies support this and show that myricetin as well as epigallocatechin-gallate (ECGC) act as strong coalvent poisons, quercetin acts as a

**anthocyanidins act as Top2 poisons and trigger illegitimate DNA** 

A catalytic Top2 inhibitor such as dexrazoxane acts to prevent DNA from binding to Top2 thus preventing any part of the catalytic cycle to occur [55–59]. By contrast, some chemicals including bioflavonoids act as Top2 "poisons" (**Figure 5**) [28, 86]. A Top2 poison acts on Top2 after DNA binding and prevents the normal function of Top2 (step 2 of catalytic cycle, see **Figure 2**). Top2 poisons can be further classified as covalent or traditional poisons. The potential as a covalent or traditional poison is dependent on biochemical structure. These groups are not mutually exclusive and

mechanism may lead to Top2 trapping on DNA [38].

**4.1 Bioflavonoids as covalent Top2 poisons**

**repair mechanisms**

*DOI: http://dx.doi.org/10.5772/intechopen.95453*

ability to induce DNA cleavage [78].

*DNA Damage and Repair Mechanisms Triggered by Exposure to Bioflavonoids and Natural… DOI: http://dx.doi.org/10.5772/intechopen.95453*

used as a spice in eastern cooking and a known Top2 inhibitor 1,4-benzoquinone showed structural and functional similarity between the two compounds and the ability to induce DNA cleavage [78].

Triterpenoids are present in plants, widely distributed within the root, stem, leaves, bark. They are components in the waxy covering of fruits and herbs such as jujube, lavender, and thyme [79]. Triterpenoids have two major components, C5 units and isopentyl diphosphate [80], and are generally present as saponins that act as defense chemicals for protection against microbes. Triterpenoids betulin lupane and oleanane from the bark of *Phyllanthus flexuosus*, derivatives of betulinc acid, and oxygenated derivatives of oleanane called celastroloids were reported to act as human Top2 inhibitors to varying degrees [81–84]. In addition, betulinic acid which is an oxidative derivative of betullin inhibits cell proliferation by inhibiting topoisomerase-DNA binding and suppressing NF-κB activation [83].

Halogenated compounds in household and baby products include polychlorinated biphenyls (PCBs), detectable in indoor carpets, and polybrominated diphenyl ethers (PBDEs), used as flame retardants, increase DNA cleavage by TopIIα in vitro and in cultured human cells [85]. Recent CRISPR-Cas9 screening against a large panel of genotoxic agents identified the synthetic small molecule pyridostatin as a Top2 inhibitor. Pyridostatin is a G-quadruplex stabilizer and this stabilization mechanism may lead to Top2 trapping on DNA [38].
