**5. Pleiotropic effects of bioflavonoids**

Due to their antioxidant capacity, bioflavonoids are included in dietary supplements 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 lead to protein level changes, cell cycle stalling, and apoptosis [16, 69].

#### **5.1 Bioflavonoids and signal transduction pathways**

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 proinflammatory 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) pathway inducing apoptosis in pancreatic cells [109].

*DNA - Damages and Repair Mechanisms*

**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

*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.*

**4.3 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].

**84**

binding site [29].

**Figure 5.**

#### **5.2 Bioflavonoids and epigenetic modifications**

Studies in cancer cell lines demonstrate epigenetic modifications caused by bioflavonoids. Genistein, quercetin, curcumin, EGCG, hesperidin, and naringin are inhibitors of DNA methyltransferases leading to hypomethylation of DNA. In addition, many of these bioflavonoids have also been shown to act on histone acetyltransferases and histone deacetyltransferases causing cell wide alterations in histone epigenetic modification patterns [109].

Long-term epigenetic effects of bioflavonoids compounds were addressed in several mouse model studies. Exposure to genistein through maternal diet during pregnancy can have long-lasting effects on the progeny. In agouti mouse pups exposed to genistein from conception until birth, epigenetic changes were observed as altered coat color, as well as significant downregulation of genes involved in hematopoiesis of bone marrow cells, increased erythropoiesis, and a permanent signature hypermethylation of repetitive elements in hematopoietic lineages [110]. Likewise, in mice exposed to quercetin from conception until birth resulted in upregulated iron-associated cytokine expression, significantly increased iron storage in the liver, and hypermethylation of repetitive elements. Epigenetic modifications lead to long term gene expression changes of cytokines associated with inflammation in the liver of the mice in adulthood [111, 112].

## **6. Implications for human health**

#### **6.1 Potential anti-cancer applications**

While bioflavonoids can be beneficial through intake at low or moderate doses, high doses and acute exposure of bioflavonoids may more drastically inhibit Top2 and impact genome integrity and cell survival, thus changing their overall impact on cells and human health. *In vitro* studies support the idea that bioflavonoids genistein and quercetin may act as chemo-preventive or anti-cancer agents by altering major processes within cancer cells such as apoptosis, cell cycle, angiogenesis and metastasis [113, 114]. Genistein has synergistic behavior with well-known anticancer drugs adriamycin, docetaxel, and tamoxifen, suggesting a potential role in combination cancer therapy [78]. Quercetin in combination with doxorubicin was found to be more effective in inducing apoptosis within the SKOV-3 cells [114]. A combinatory treatment with quercetin and curcumin synergistically induce anticancer activity in triple-negative breast cancer cells by modulating tumor suppressor genes in particular enhancing BRCA1 expression [115].

Several bioflavonoids have been investigated as alternate cancer therapeutics that are less genotoxic than traditional chemotherapeutics but equally effective. High concentrations of myricetin causes Top2-mediated DNA damage and apoptosis in K652 cells [116]. Fisetin interrupts the MAPK-dependent NF-κB signaling pathway in cervical cancer cells, inhibiting migration and invasion [114]. Several *in vitro* and *in vivo* studies indicate that luteolin can suppress metastasis of breast cancer by reversing epithelial-mesenchymal transition, or by acting as an antiangiogenic therapeutic inhibiting VEGF production and suppressing invasion [117, 118].

While these observations strengthen the notion that flavonoids could be useful anti-cancer agents, to date minimal clinical studies have demonstrated that these bioflavonoids retain anti-cancer properties in humans *in vivo*. A Phase I study/ pharmacokinetic trial of quercetin in cancer patients intravenously injected quercetin in 11 patients with cancer at varying doses of 60–2000 mg/m<sup>2</sup> and identified 945 mg/m<sup>2</sup> as a safe and effective dose [119].

**87**

chemotherapeutic agents.

**7. Conclusion**

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

Aberrant repair of DNA DSBs caused by either endogenous or exogenous agents has the potential to result in DNA sequence mutations or genome rearrangements such as chromosomal translocations which can lead to disease. Negative consequences of high bioflavonoid intake can be observed most prominently in pregnant women. Epidemiological data from countries whose citizens have higher bioflavonoid intake (particularly soy products) had a 2–3 times higher incidence of infant leukemia, characterized by chromosomal translocation, suggesting maternal intake of high amounts of bioflavonoids could lead to this particular genome rearrange-

Infant leukemia typically occurs due to translocation events involving the mixed lineage leukemia (*MLL*) gene. Most of the *MLL* rearrangements observed in patients with infant leukemia and therapy-related leukemia (tAML) cluster together in a well-defined region of the *MLL* locus [121]. tAML is associated with treatment with Top2 poisons etoposide or doxorubicin [86, 88, 121] which has led to the hypothesis and working model that ingestion of natural Top2 poisons including bioflavonoids can lead to these translocation events and tumorigenesis [121, 122]. In support of this, boflavonoids have been shown to inhibit Top2 and induce *MLL* cleavage and

translocations in hematopoietic stem cell-enriched populations [87, 121].

Foods contain multiple different bioflavonoids, and bioflavonoids are bioaccumulative which likely increases plasma concentrations [123]. Study of the potential for environmental or dietary compounds to induce infant leukemias is more relevant since they cross the placental barrier as shown with the synthetic bioflavonoid EMD-49209 [124], genistein [111, 125], quercetin [111], herbal medicines, dipyrone, and pesticides including the mosquitocidal Baygon [126, 127]. Genotoxic effects of quercetin on the human hemopoietic stem and progenitor cells (HSPCs) were shown using a genetically engineered placental barrier model from a specialized human cell line. This study showed that approximately 10% of quercetin from the maternal side is capable of crossing the placental barrier and accumulating in the fetus. Exposure *in utero* is likely more damaging due to differences in metabolic and excretion rates of mother and fetus [128] as well as rapidly developing and proliferating fetal cells that are more sensitive to Top2 inhibiting agents [129].

Bioflavonoids are prevalent in the human diet from natural sources such as fruits and vegetables, but are also found at supranatural concentrations in dietary supplements and energy drinks. These chemical compounds have numerous cellular effects including interfering with signal transduction pathways, modifying the DNA damage response and epigenetic markers, and poisoning of Top2 causing DNA DSBs and leading to aberrant repair. Given the number of cellular pathways bioflavonoids affect, and the DNA damage caused by bioflavonoid exposure, it is possible that bioflavonoids could be used as natural analogs of traditional chemotherapeutic agents. However, more research is needed to understand how these bioflavonoids cause DNA damage through Top2-dependent or -independent pathways to understand potential off-target negative effects. In addition, further research will be needed to understand the dose-dependent activities of bioflavonoids and at what doses they may be chemo-protective versus what threshold doses they may induce DNA damage that is mutagenic, and finally at what high acute doses they may induce DNA damage and apoptosis to act as effective alternative to traditional

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

**6.2 Potential inducers of infant leukemia**

ment and infant leukemia [120].

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

### **6.2 Potential inducers of infant leukemia**

*DNA - Damages and Repair Mechanisms*

**5.2 Bioflavonoids and epigenetic modifications**

histone epigenetic modification patterns [109].

of the mice in adulthood [111, 112].

**6. Implications for human health**

**6.1 Potential anti-cancer applications**

sor genes in particular enhancing BRCA1 expression [115].

cetin in 11 patients with cancer at varying doses of 60–2000 mg/m<sup>2</sup>

as a safe and effective dose [119].

Studies in cancer cell lines demonstrate epigenetic modifications caused by bioflavonoids. Genistein, quercetin, curcumin, EGCG, hesperidin, and naringin are inhibitors of DNA methyltransferases leading to hypomethylation of DNA. In addition, many of these bioflavonoids have also been shown to act on histone acetyltransferases and histone deacetyltransferases causing cell wide alterations in

Long-term epigenetic effects of bioflavonoids compounds were addressed in several mouse model studies. Exposure to genistein through maternal diet during pregnancy can have long-lasting effects on the progeny. In agouti mouse pups exposed to genistein from conception until birth, epigenetic changes were observed as altered coat color, as well as significant downregulation of genes involved in hematopoiesis of bone marrow cells, increased erythropoiesis, and a permanent signature hypermethylation of repetitive elements in hematopoietic lineages [110]. Likewise, in mice exposed to quercetin from conception until birth resulted in upregulated iron-associated cytokine expression, significantly increased iron storage in the liver, and hypermethylation of repetitive elements. Epigenetic modifications lead to long term gene expression changes of cytokines associated with inflammation in the liver

While bioflavonoids can be beneficial through intake at low or moderate doses, high doses and acute exposure of bioflavonoids may more drastically inhibit Top2 and impact genome integrity and cell survival, thus changing their overall impact on cells and human health. *In vitro* studies support the idea that bioflavonoids genistein and quercetin may act as chemo-preventive or anti-cancer agents by altering major processes within cancer cells such as apoptosis, cell cycle, angiogenesis and metastasis [113, 114]. Genistein has synergistic behavior with well-known anticancer drugs adriamycin, docetaxel, and tamoxifen, suggesting a potential role in combination cancer therapy [78]. Quercetin in combination with doxorubicin was found to be more effective in inducing apoptosis within the SKOV-3 cells [114]. A combinatory treatment with quercetin and curcumin synergistically induce anticancer activity in triple-negative breast cancer cells by modulating tumor suppres-

Several bioflavonoids have been investigated as alternate cancer therapeutics that are less genotoxic than traditional chemotherapeutics but equally effective. High concentrations of myricetin causes Top2-mediated DNA damage and apoptosis in K652 cells [116]. Fisetin interrupts the MAPK-dependent NF-κB signaling pathway in cervical cancer cells, inhibiting migration and invasion [114]. Several *in vitro* and *in vivo* studies indicate that luteolin can suppress metastasis of breast cancer by reversing epithelial-mesenchymal transition, or by acting as an antiangiogenic therapeutic inhibiting VEGF production and suppressing invasion [117, 118]. While these observations strengthen the notion that flavonoids could be useful anti-cancer agents, to date minimal clinical studies have demonstrated that these bioflavonoids retain anti-cancer properties in humans *in vivo*. A Phase I study/ pharmacokinetic trial of quercetin in cancer patients intravenously injected quer-

and identified

**86**

945 mg/m<sup>2</sup>

Aberrant repair of DNA DSBs caused by either endogenous or exogenous agents has the potential to result in DNA sequence mutations or genome rearrangements such as chromosomal translocations which can lead to disease. Negative consequences of high bioflavonoid intake can be observed most prominently in pregnant women. Epidemiological data from countries whose citizens have higher bioflavonoid intake (particularly soy products) had a 2–3 times higher incidence of infant leukemia, characterized by chromosomal translocation, suggesting maternal intake of high amounts of bioflavonoids could lead to this particular genome rearrangement and infant leukemia [120].

Infant leukemia typically occurs due to translocation events involving the mixed lineage leukemia (*MLL*) gene. Most of the *MLL* rearrangements observed in patients with infant leukemia and therapy-related leukemia (tAML) cluster together in a well-defined region of the *MLL* locus [121]. tAML is associated with treatment with Top2 poisons etoposide or doxorubicin [86, 88, 121] which has led to the hypothesis and working model that ingestion of natural Top2 poisons including bioflavonoids can lead to these translocation events and tumorigenesis [121, 122]. In support of this, boflavonoids have been shown to inhibit Top2 and induce *MLL* cleavage and translocations in hematopoietic stem cell-enriched populations [87, 121].

Foods contain multiple different bioflavonoids, and bioflavonoids are bioaccumulative which likely increases plasma concentrations [123]. Study of the potential for environmental or dietary compounds to induce infant leukemias is more relevant since they cross the placental barrier as shown with the synthetic bioflavonoid EMD-49209 [124], genistein [111, 125], quercetin [111], herbal medicines, dipyrone, and pesticides including the mosquitocidal Baygon [126, 127]. Genotoxic effects of quercetin on the human hemopoietic stem and progenitor cells (HSPCs) were shown using a genetically engineered placental barrier model from a specialized human cell line. This study showed that approximately 10% of quercetin from the maternal side is capable of crossing the placental barrier and accumulating in the fetus. Exposure *in utero* is likely more damaging due to differences in metabolic and excretion rates of mother and fetus [128] as well as rapidly developing and proliferating fetal cells that are more sensitive to Top2 inhibiting agents [129].
