**4. Flavonols, flavonols, flavones, isoflavones, flavanones, and anthocyanidins act as Top2 poisons and trigger illegitimate DNA repair mechanisms**

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

#### **4.1 Bioflavonoids as covalent Top2 poisons**

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

*DNA - Damages and Repair Mechanisms*

reproduction of other plants [60].

signal transduction pathways [69].

**3.4 Additional compounds as Top2 inhibitors**

**3.3 Flavonols**

athletes [72, 73].

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

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

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

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].

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

**82**

#### **Figure 5.**

*Bioflavonoid classification as a covalent or traditional topoisomerase II poison. The blue boxed regions indicate required biochemical features for a traditional Top2 poison. The red 3′, 4′ and 5'-OH groups on the B ring are necessary for covalent Top2 poisons. The 4'-OH group is required for covalent binding, while the 3′, 5′ increase the binding affinity, therefore a bioflavonoid with all 3-OH groups would be a strong covalent poison.*

#### **4.2 Bioflavonoids as traditional Top2 poisons**

Flavones, flavonols, isoflavones, and flavanones (but not flavanols or anthocyanidins) have the all act as traditional (or interfacial) Top2 poisons. The key structural components for a traditional Top2 poison are a 5'-OH group in the A ring, a 4'-OH group in the B ring, and a 4′ = O in the C ring (**Figure 5**). A traditional (or interfacial) Top2 poison stalls the enzyme by binding to the active site of the enzyme preventing religation, thereby resulting in the formation of a stabilized cleavage complex (SCC) [88, 89]. Flavonols are strong traditional poisons and both cell free and cell culture systems support this. Similarly, experiments in cell culture systems examining the kinetics of DSB repair following exposure to acute doses of bioflavonoids support the model that flavonols, flavones, and isoflavones including kaempferol, quercetin, myricetin, genistein, and luteolin and each act as a traditional Top2 poison. However, combinatorial activity of genistein, quercetin and luteolin together suggests they may have weak covalent poisoning capabilities when they have to compete for the traditional poisoning binding site [29].

#### **4.3 Bioflavonoids trigger illegitimate DNA repair mechanisms**

Bioflavonoids with either covalent and traditional Top2 poisoning activity induce the DSB-mediated DDR as evidenced by induction of γ-H2AX foci, ATM phosphorylation, and p53 signaling [90–92]. However, a more direct role or influence of these compounds on the repair of damage is not as clear [93]. Acute doses induce DNA damage and DDR as detected by γ-H2AX foci and phosphorylation of ATM in stem cells and CD34+ hematopoietic progenitor cells [94, 95].

**85**

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

Genistein and quercetin inhibit Top2 to induce DNA DSBs, and also appear to influence DSB repair pathway choice. Protein level analysis for HR, C-NHEJ, and Alt-EJ specific proteins suggests that genistein and quercetin suppress HR by reducing BRCA2 and Rad51 expression, as well as suppress C-NHEJ by suppressing levels of DNA-PKcs, Ku80, XLF and XRCC4 and trigger Alt-EJ by increasing levels of CtIP and Polymerase θ [96, 97]. DNA reporter assays suggest that quercetin interferes with DNA repair mechanisms such as HR and C-NHEJ by inhibition of PI3K/Akt signaling. In support of these studies, exposure to multiple bioflavonoids promotes the generation of chromosomal translocations in a

Bioflavonoids that have traditional Top2 poisoning activity lead to trapped SCCs on the DNA. Removal of SCCs is performed by the small ubiquitin-related modifier ligase ZNF45/tyrosyl-DNA phosphodiesterase 2 (ZATT/TDP2) complex. Removal of the SCC is required for DSB repair by C-NHEJ. If ZATT/TDP2 does not remove the SCC, the MRN complex or CtIP with nuclease activity may resect the DNA ends with the SCC attached to allow for DSB repair by HR or Alt-EJ [55, 94, 98–101]. Inhibition or mutation of multiple DNA repair proteins potentiates cytotoxicity of Top2 inhibitors, and MRE11 plays a direct mechanistic role in removal of Top2-DNA

Due to their antioxidant capacity, bioflavonoids are included in dietary supple-

ments for their presumed health benefits in protecting against inflammation, cardiovascular diseases, and cancer [87]. These beneficial health properties are due to the number of pleiotropic effects bioflavonoids have on cells by impacting signal transduction pathways, DSB repair and the cellular epigenetic landscape, which can

Bioflavonoids have antioxidant and anti-inflammatory properties. Their antioxidant properties are due to their ability to reduce reactive oxygen species of the multiple –OH groups in their chemical structure. Their anti-inflammatory properties are due to their interference with signal transduction pathways and down-regulation in the production of pro-inflammatory cytokines. Bioflavonoids decrease inflammation and immune cell recruitment through interference with the ERK/MAP kinase and NF-κB signal transduction pathways which can be beneficial to human health. NF-κB is a transcription factor that upon activation is transported into the nucleus and binds to the promoter region for a number of cytokines and apoptotic genes; therefore reduced pathway activation leads to lower pro-inflammatory cytokine production and increased cell survival [104]. Extracts from the plant *Ginkgo biloba*, rich in bioflavonoids, act as an herbal antioxidant, augment the transcription of TNF-α causing reduced activation of the NF-κB pathway. Apigenin has shown similar down regulatory effects on cytokine production likely through the modulation of NF-κB activation [105]. Quercetin and fisetin inhibit pro-

inflammatory cytokine production through the suppression of NF-κB activation by decreased phosphorylation of extracellular signal-regulated (ERK) kinase and p38 mitogen-activated protein (MAP) kinase that are activators of NF-κB [106–108]. Myricetin has been shown to affect the phosphatidylinositol 3-kinase (PI3-K)

lead to protein level changes, cell cycle stalling, and apoptosis [16, 69].

**5.1 Bioflavonoids and signal transduction pathways**

pathway inducing apoptosis in pancreatic cells [109].

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

dose-dependent manner [29, 87].

complexes in yeast and mammals [102, 103].

**5. Pleiotropic effects of bioflavonoids**

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

Genistein and quercetin inhibit Top2 to induce DNA DSBs, and also appear to influence DSB repair pathway choice. Protein level analysis for HR, C-NHEJ, and Alt-EJ specific proteins suggests that genistein and quercetin suppress HR by reducing BRCA2 and Rad51 expression, as well as suppress C-NHEJ by suppressing levels of DNA-PKcs, Ku80, XLF and XRCC4 and trigger Alt-EJ by increasing levels of CtIP and Polymerase θ [96, 97]. DNA reporter assays suggest that quercetin interferes with DNA repair mechanisms such as HR and C-NHEJ by inhibition of PI3K/Akt signaling. In support of these studies, exposure to multiple bioflavonoids promotes the generation of chromosomal translocations in a dose-dependent manner [29, 87].

Bioflavonoids that have traditional Top2 poisoning activity lead to trapped SCCs on the DNA. Removal of SCCs is performed by the small ubiquitin-related modifier ligase ZNF45/tyrosyl-DNA phosphodiesterase 2 (ZATT/TDP2) complex. Removal of the SCC is required for DSB repair by C-NHEJ. If ZATT/TDP2 does not remove the SCC, the MRN complex or CtIP with nuclease activity may resect the DNA ends with the SCC attached to allow for DSB repair by HR or Alt-EJ [55, 94, 98–101]. Inhibition or mutation of multiple DNA repair proteins potentiates cytotoxicity of Top2 inhibitors, and MRE11 plays a direct mechanistic role in removal of Top2-DNA complexes in yeast and mammals [102, 103].
