Dietary Flavonoids: Effects on the Gene Expression And

DIETARY FLAVONOIDS: EFFECTS ON THE GENE EXPRESSION AND

ACTIVITY OF NQO1

A Project

Presented to the

Faculty of

California State Polytechnic University, Pomona

In Partial Fulfillment

Of the Requirements for the Degree

Master of Science

Nutrition and Food Science

Gianluis Pimentel-Barrantes

2020 SIGNATURE PAGE

PROJECT: DIETARY FLAVONOIDS: EFFECTS ON THE GENE EXPRESSION AND ACTIVITY OF NQO1

AUTHOR: Gianluis Pimentel-Barrantes

DATE SUBMITTED: Summer 2020 Department of Nutrition and Food Science

Erik Froyen, Ph.D Project Committee Chair Nutrition and Food Science

Valerie Mellano, Ph.D Plant Science

Gabriel Davidov-Pardo, Ph.D Nutrition and Food Science

ii ACKNOWLEDGEMENTS

Foremost, I would like to thank Jesus, my God in heaven, for guiding me and giving me the strength to complete this project. I would not have been able to do it without Him. I would also like to express my sincere gratitude to my advisor Dr. Erik

Froyen for the continuous support. Thank you for sharing your immense knowledge and for being a humble, motivational example. I would also like to thank the rest of my committee: Dr. Davidov-Pardo and Dr. Valerie Mellano for their encouragement, insightful comments, and patience throughout the process.

I would like to thank my family, especially my brother Hector Alain for his continuous support throughout my studies. I would not have been able to do this without his support and I will always be grateful for what he has done for me. Finally, I want to thank my father Hector and my mother Marialuisa for supporting me to pursue an education in America and for being such a great example.

iii ABSTRACT

It is important to investigate which flavonoids are involved in the metabolism of xenobiotics to ameliorate the incidence of cancer around the world. Populations around the world are constantly exposed to a diversity of pollutants from the air, water, and soil.

Substantial scientific evidence demonstrates that flavonoid-rich diets that are abundant in a diversity of fruits and vegetables can protect eukaryotic cells from oxidative stress. It has been found that multiple flavonoids from various subclasses such as anthocyanidins

(anthocyanin, cyanidin, pelargonidin, kuromanin, delphinidin, malvidin), flavan-3-ols

(epigallocatechin gallate, catechin), flavonols (quercetin, kaempferol, galangin, isorhamnetin, myricetin, morin, taxifolin, hyperoside, rutin), flavones (apigenin, diosmetin, luteolin, scutellarin, rutin, 4’dimethylnobiletin), and isoflavones (genistein, daidzein, equol, biochanin A, cuomestrol, tectorigenin, glycitein) can modulate quinone reductase (QR) activity and mRNA expression in cell and animal studies and therefore increase the excretion of carcinogens. Although, more research needs to be done on the mechanisms by which flavonoids induce QR to be able to provide precise dietary recommendations of flavonoids to humans. This paper presents the current knowledge of single isolated flavonoids and their roles in xenobiotic metabolism.

iv TABLE OF CONTENTS

SIGNATURE PAGE...... ii

ACKNOWLEDGMENTS ...... iii

ABSTRACT...... iv

CHAPTER 1: Introduction ...... 1

CHAPTER 2: Methodology...... 5

CHAPTER 3: Results ...... 6

CHAPTER 4: Discussion...... 10

CHAPTER 5: Conclusion...... 19

REFERENCES ...... 20

APPENDIX: Tables & Figures...... 33

v CHAPTER 1: Introduction

The prevalence of cancer is among the leading causes of death worldwide, and the number of cases per year is expected to rise to 23.6 million by 2030. National Cancer

Institute (2018) explained the incidence of cancer has become a burden to the American society, as it was estimated that in 2018 a total of 1,735,350 new cases were diagnosed.

The estimated national expenditure for cancer care has dramatically increased over the years, as in 2017 in the United States $147.3 billion were spent. Multiple epidemiological studies have suggested that diets rich in fruits and vegetables may protect humans from developing chronic diseases such as cancer, cardiovascular disease, and diabetes. Studies have demonstrated that certain phytochemicals which are abundant in plants, can induce the expression of the phase II enzymes in the liver responsible for xenobiotic metabolism

(Dinkova-Kostova et al., 2004). The process of xenobiotic metabolism consists of enzymatic reactions which transforms chemicals such as carcinogens and drugs into forms readily for excretion (Ierapetritou et al., 2009). It is crucial to understand the pharmacological roles of phytochemicals in xenobiotic metabolism, as humans today are exposed to a vast diversity of contaminants (Barradas-Gimate et al., 2017).

Electrophiles and oxidants such as quinone metabolites, are ubiquitous in aerobic organisms such as plants and mammals as a result from metabolic processes and xenobiotic metabolism (Raymond & Segre, 2006). However, exogenous sources of quinone metabolites can also be derived from the burning of fossil fuels which generate the polycyclic aromatic hydrocarbons (PAHs), the intake of drugs, exposure to pesticides and other similar activities commonly seen in industrialized nations (Monks & Jones,

2002). For instance, α- and β-unsaturated aldehydes byproducts from lipid peroxidation

1 and industrial processes are harmful electrophiles that have the capacity to promote cellular oxidative stress; thus, disrupting DNA, proteins, and plasma membranes (Bossy-

Wetzel et al., 2004). A large body of evidence reviewed in Braeuning et al. (2006) confirms that frequent cellular exposure to free radicals, promotes the synthesis of reactive oxygen species that facilitate the development of chronic diseases such as different types of cancers, cardio vascular disease, atherosclerosis, chronic inflammatory disease, faster aging, and neurodegenerative conditions like Alzheimer’s disease. The

National Toxicology Program from the U.S. Department of Health and Human Services has reported a list of 248 harmful substances that promote toxicity and development of cancer in humans based on the available scientific evidence. Common examples include

PAHs, halogenated aromatic hydrocarbons, and heterocyclic amines (Nebert et al., 2004).

The major source of heterocyclic amines in the United States comes from barbeque meats, as amino acids are converted into these products from cooking meats under high temperatures (Froyen EB, 2016; Sinha et al., 1998).

Recent studies have demonstrated that a diversity of compounds such as phytochemicals, endogenous chemicals, therapeutics, environmental agents, and exogenous chemicals (electrophiles) modulate the ARE/EpRE gene involved in the transcription of phase II enzymes (Dinkova-Kostova et al., 2004). For instance, some of the hydroxylated flavonoids ubiquitously found in plants, have been noted to modulate cell signaling cascades. However, among the 12 subclasses of flavonoids, only 6 have been marked of dietary significance which are anthocyanidins, flavan-3-ols, flavonols, flavones, flavanones, and isoflavones (Table 1 & Figure 1) (Higdon et al., 2016). Studies have shown that the activation of electrophile-responsive element (ARE/EpRE) may

2 occur via electrophiles and other compounds at the promoter region (Lee & Surh, 2005).

The activation of EpRE occurs via the family of transcription factors nuclear factor erythroid 2–related factor 2 (Nrf2) binding to EpRE. The transcription factor Nrf2 is a member of the cap ‘n’ collar subfamily of basic region leucine zipper transcription factors which are in the cytosol under resting conditions (Moi et al., 1994). When Nrf2 is found in the cytosol is normally bound to the cytoskeleton-bound Kelch-like erythroid cell-derived protein with cap ‘n’ collar homology–associated protein 1 (Keap1) (Zhang et al., 2009). The entry of environmental carcinogens or phytochemicals into the cytosol, promotes the dissociation between Nrf2 and Keap1, allowing the Nrf2 to translocate into the nucleus to promote the transcription of phase II enzymes (Nguyen et al., 2004;

Kobayashi & Yamamoto, 2005).

Nrf2 has been identified to mediate the induction of phase II enzymes such as:

NADPH-dependent quinone oxidoreductase-1 (NQO1), heme-oxygenase 1 heat shock protein 32 (HO-1), glutathione-S-transferase (GST), DP-glucuronosyltransferase 1A6, and sulfurotransferases (Aleksunes et al., 2006; Itoh et al., 1997). In addition, different kinases enzymes are involved in the activation of Nrf2, such as mitogen-activated protein kinase (MAPK), protein kinase C, and phosphatidylinositol 3-kinase (PI3K) (Eggler et al., 2008). The MAPK family of kinases includes the JNK, ERK, and p38 which are regulators of downstream transcription factors involved in carcinogenesis mechanisms such as proliferation, differentiation, apoptosis, and tumor induction (Bak et al., 2012). In addition, the MAPK family of kinases are involved in the activation of ARE genes that mediate the transcription of the phase II family enzymes (Figure 3) (Yu et al., 2000).

3 The enzyme NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) also known as quinone reductase (QR), is a phase II enzyme that uses NADH or NAD(P)H as an electron donor to reduce quinones to hydroquinones (Lin et al., 2017). This enzyme is capable to increase the polarity of a carcinogen, to facilitate its excretion from the body.

NQO1 is a flavoprotein that catalyzes the two-electron reduction of quinones without forming free radicals along the way (Riley & Workman, 1992). NQO1 utilizes flavin adenine dinucleotide (FAD) and dihydronicotinamide-adenine dinucleotide phosphate

(NAD(P)H) as cofactors to undergo its enzymatic mechanisms. NQO1 can also reduce chromium toxicity and detoxify benzo(α)pyrene (Joseph & Jaiswal, 1994). This enzyme supplies the necessary tools for cells to reduce potentially detrimental compounds to less reactive metabolites that may be eliminated via the liver, gut, kidney, or lungs (Almazroo et al., 2017). In addition, oxidoreductases have been reported to successfully act on organic substrates including amines, ketones, alcohols, as well as inorganic substrates such as heavy metals and sulfites (Vidal et al., 2018). The QR assay is based on the production of a blue color when MTT is reduced nonenzymatically by menadiol that is generated enzymatically from menadione by quinone reductase (Figure 2) (Prochaska &

Santamaria, 1988).

The specific aim of this paper was to look at the effects of flavonoids on the gene expression and activity of QR in cell and animal studies. It will be beneficial to look at which flavonoids are involved in the metabolism of xenobiotics to decrease the progression of chronic diseases like cancer. This paper also provides insight into our comprehension of flavonoids as modulators of QR, since there has not been any comprehensive reviews on this topic.

4 CHAPTER 2: Methodology

To complete this paper, articles that investigated the effects of flavonoids on gene

expression and activity of QR were searched on Pubmed in May 2020. The following key

words were used to search for primary research articles that included commonly

consumed flavonoids: catechin nqo1, malvidin nqo1, epigallocatechin nqo1, isorhamnetin

nqo1, myricetin nqo1, quercetin nqo1, apigenin nqo1, luteolin nqo1, eriodicytol nqo1,

hasperetin nqo1, naringenin nqo1, daidzein nqo1, genistein nqo1, genistein qr, glycitein

nqo1, biochanin a qr, formononetin qr, anthocyanidins nqo1, flavonols nqo1, flavones

nqo1, anthocyanidins nqo1, flavanols nqo1, isoflavones nqo1, morin nqo1, and

pinostrobin qr (Higdon et al., 2005). Only articles that utilized isolated flavonoids were

included in this paper. Articles that were excluded for not meeting the criteria were studies that included a mix of various phytochemicals such as extracts, herbs, or concentrates.

5 CHAPTER 3: Results

3.1 Modulation of NQO1 via Anthocyanidins in Cell Studies

The modulation of the phase II enzyme NQO1 was reported to be affected by

anthocyanidins in cell studies (Table 2). Anthocyanin and cyanidin increased NQO1

protein level in human colorectal adenocarcinoma and human neuroblastoma cells (Ma et

al., 2019; Thummayot et al., 2018). Quinone reductase (QR) activity and protein levels of

NQO1 were increased by cyanidin, kuromanin, delphinidin, and malvidin in rat liver

(Clone-9) cells (Table 3) (Shih et al., 2007). Li et al. (2019) reported an increase of

NQO1 protein level and mRNA expression by pelargonidin in mouse skin epidermal

cells.

3.2 Modulation of NQO1 via Flavan-3-ols in Cell and Animal Studies

A number of flavan-3-ols exert the gene induction of NQO1 in cell and animal studies (Table 3 & Table 8). Treatment with epigallocatechin gallate and catechin in mouse hepatoma (Hepa-1c1c7) and human primary skin fibroblast (142Br) cells induced the mRNA expression of NQO1 (Muzolf-Panek et al., 2008; Warwick et al., 2012).

Protein levels and mRNA expression of NQO1 by treatment with (-)-epigallocatechin-3- gallate were increased in Kunming mice. However, catechin was reported to decrease QR activity and NQO1 mRNA expression in Wistar Unilever rats (Table 3) (Wang et al.,

2015; Heike et al., 2009).

3.3. Modulation of NQO1 via Flavonols in Cell and Animal Studies

Multiple flavonols modulated the activity and gene expression of NQO1 in cell and animal studies (Table 4 & Table 9). The flavonol quercetin increased mRNA expression of QR in human retinal pigment epithelial (ARPE-19), human hepatocyte

6 (HepG-2), human colon carcinoma (Cacao-2) cells; however, no change was reported in human primary skin fibroblast (142 Br) cells (Xu et al., 2016; Taniwaga et al., 2007;

Niestroy et al., 2011; Warwick et al., 2012). Quercetin and dihydroquercetin increased

NQO1 protein level in human breast carcinoma (MCF-7), ARPE-19, and HepG-2 cells, while only quercetin affected QR activity in Hepa-1c1c7 cells (Valerio et al., 2001; Weng et al., 2017; Liang et al., 2013; Uda et al., 1997). Protein levels and mRNA expression of

NQO1 were increased by quercetin in C57BL/6J mice and broiler chickens (Maturu et al., 2018; Wei et al., 2017; Sun et al., 2020); while only increasing mRNA expression in

C57BL/6J (Vanhees et al. 2012). No change in QR activity nor mRNA expression by quercetin was observed in Wistar Unilever rats (Heike et al., 2009). Treatment with kaempferol increased mRNA expression of NQO1 in Caco-2 cells (Niestroy et al., 2011) and increased QR activity in Hepa-1c1c7 cells (Uda et al. 1997). Galangin increased QR activity in Hepa-1c1c7 and isorhamnetin increased protein levels in HepG-2 cells (Uda et al. 1997; Yang et al., 2014). Taxifolin, hyperoside, and rutin increased protein levels and mRNA expression of NQO1 in ARPE-19, human kidney proximal tubular cells (HK-2), and HepG-2 cells (Xie et al., 2017; Chen et al., 2017; Karakurt et al., 2016). Protein levels and mRNA expression of QR were increased by myricetin and dihydromyricetin in

Sprague Dawley rats (Chu et al., 2018; Sang et al., 2017), while only increasing mRNA expression in C57BL/6J (Zhang et al., 2016). QR activity was reported to increase by morin in Fischer 344 albino rat (F344) (Kawabata et al., 1999; Tanaka et al., 2018).

3.4 Modulation of NQO1 via Flavones in Cell and Animal Studies

Various flavones induced gene expression of NQO1 in cell and animal studies

(Table 5 & Table 10). The protein levels of NQO1 were increased by luteolin, apigenin,

7 apigetrin, scutellarin, and 4’dimethylnobiletin in HepG2, normal human bronchial epithelial cells (NHBE), Sprague Dawley, Wistar Unilever, Kunming mouse, C57BL/6J and CD-1 mice (Kitakaze et al., 2019; Tan et al., 2014; Tan et al., 2020; Tan et al., 2018;

Yang et al., 2016; Yang et al., 2018; Guo et al., 2019; Fan et al., 2017;Wu et al., 2015).

Apigenin, diosmetin, and luteolin induced NQO1 protein levels and mRNA expression in mouse skin epidermal cells (JB6-6P), human normal hepatocyte cells (L02), C57BL/6J, and ICR mice (Paredes-Gonzalez et al., 2014; Wang et al., 2018; Li et al., 2016; Xu et al.,

2014). Both apigenin and rutin increased NQO1 mRNA expression in ARPE-19 and

Wistar Unilever respectively; however, luteolin decreased it in HepG-2 cells (Xu et al.,

2016; Zhang et al., 2014; Balakina et al., 2016). Khan et al. (2006) reported an increased in protein level of NQO1 by apigenin in Swiss Webster.

3.4 Modulation of NQO1 via Flavanones in Cell and Animal Studies

Flavones were found to modulate the gene expression of NQO1 cell and animal studies (Table 6 & Table 11). The mRNA expression of NQO1 was increased by hesperidin, naringenin, and naringenin-7-0-glucoside in rat cardiac cells (H9C2), Wistar

Unilever, and Sprague Dawley respectively (Han et al., 2008; Balakina et al., 2016;

Esmaeili et al., 2014). Eriodicytol, hesperetin, and naringenin increased NQO1 protein levels in ARPE-19, HK-2, mouse insulinoma cells (MIN-6), and Sprague Dawley respectively (Johnson et al., 2009; Chen et al., 2019; Rajappa et al., 2019; Kapoor et al.,

2019). Fahey et al. (2002) reported an increase in QR activity by pinostrobin in Hepa-

1c1c7 cells.

8 3.5 Modulation of NQO1 via Isoflavones in Cell and Animal Studies

Certain isoflavones up regulated the expression of NQO1 in cell and animal studies (Table 7 & Table 12). Genistein was reported to increase QR activity and mRNA expression of NQO1 in human colon cancer cells (Colo205) and Wistar Unilever (Wang et al., 1998; Wiegand et al., 2009). QR activity was increased by genistein in Hepa-

1c1c7, Swiss Webster, and Sprague Dawley (Yannai et al., 1998; Froyen et al., 2009;

Appelt & Reicks, 1999). The mRNA expression and protein level were increased by genistein in mouse breast cancer cells (MDA-MB-231) (Bianco et al., 2005). Genistein increased QR protein level and mRNA expression in 129/sv mice and Sprague Dawley respectively and it increased mRNA expression, protein level, and activity in hepa-1c1c7

(Bai et al., 2019; Miao et al., 2018; Froyen et al., 2011). Daidzein was reported to increase QR activity in Hepa-1c1c7 and Sprague Dawley, while protein levels were elevated in human umbilical vein endothelial cells (EAhy926) (Yannai et al., 1998;

Appelt et al., 1999; Zhang et al., 2013). The QR mRNA expression, activity, and protein levels were elevated by daidzein and equol in Hepa-1c1c7 (Froyen et al., 2011). Protein levels of NQO1 were elevated by S-(-) equol and biochanin A in Hepa-1c1c7 and

EAhy926 respectively (Froyen et al., 2011; Zhang et al., 2013). QR activity and mRNA expression were elevated by biochanin A and coumestrol in Colo205 (Wang et al., 1998).

Protein levels and mRNA expression of NQO1 were elevated by tectorigenin and glycitein in C6 astrogloma cells (Park et al., 2011).

9 CHAPTER 4: Discussion

4.1 QR Activity and mRNA Expression was Modulated in Various Cell Lines and

Animal Models

The results from the presented studies indicate that multiple flavonoids modulate the gene expression of the phase II detoxification enzyme NQO1, which suggests that some flavonoids may serve as key pharmacological agents to fight against diseases perpetuated by oxidative stress and toxicity in eukaryotic cells. Almost all studies utilized concentrations of flavonoids of low physiological relevance and only a few used flavonoid metabolites. The physiological concentration of flavonoids in the blood is typically less than 1 µM (Kanazawa, 2011). However, a few studies successfully demonstrated the modulation of NQO1 via increased mRNA gene expression with concentrations of ≤1 µM (Bianco et al., 2005; Froyen et al., 2011; Ma et al., 2019;

Niestroy et al., 2011; Wang et al., 1998). The studies from (Tables 8 - 12) gave no considerations on how the doses fed to animals could be applicable to human intake.

However, animal studies that have used concentrations of genistein of 1500 mg/kg diet have shown to result in serum concentrations of 1.12 µM in mice, which it is within the range of what it would result in humans after consuming soy products (Froyen &

Steinberg, 2016; Ju et al., 2006; Klein & King, 2007; Wiegand et al., 2009). The animal- model experiments that provided the flavonoid treatment via injection, is not physiologically relevant as it bypasses the metabolism of the compound as it moves through the digestive system and the liver.

Anthocyanidins can be found in food sources such as red, blue, and purple berries

(Table 1). Flavonoids from the anthocyanidin subclass which are characterized by a

10 heteroaromatic C15 three ring skeleton (Figure 1), promote numerous health-promoting properties (Sinopoli et al., 2019). Anthocyanidins such as anthocyanin, cyanidin, pelargonidin, kuromanin, delphinidin, and malvidin up-regulated NQO1 expression and increased the binding of Nrf2 to the ARE promoter region. Kuromanin, delphinidin, malvidin, pelargonidin, and anthocyanin were defined as chemopreventive blocking reagents by significantly increasing QR activity and mRNA levels (Li et al., 2019; Ma et al., 2019; Shih et al., 2007). Thummayot et al. (2018) reported that cyanidin has a great potential as a therapeutic agent in neurodegenerative diseases, as it can scavenge free radicals and up-regulate NQO1 in human neuroblastoma cells.

Flavanols can be found in various food sources such as teas, apples, berries,

(Table 1). Flavanols found in foods as either monomers such as epicatechin, or in oligomeric forms such as procyanidins, have received increasing attention due to their reported health benefits (Robbins, R. J., et al., 2006). The results from this paper indicate that epigallocatechin gallate, catechin, and (-)-epigallocatechin-3-gallate are modulators of NQO1 (Table 2 & Table 3). Warwick et al. (2012) reported a significant increase of

NQO1 mRNA in infant and adult primary skin fibroblasts, remarking the benefit of the inclusion of dietary phytochemicals in an infant’s diet, to promote protection from exposure to harmful xenobiotics later in life. However, Heike et al. (2009), reported that catechin decreased the mRNA expression of NQO1 in Wistar Unilever who were fed doses of 2 g/kg diet. The dietary flavanol concentration used in this rat study was higher than a concentration that could be obtainable in humans who consume a flavonoid-rich diet. The author however, made the remark that catechins in its purified forms are available to humans often in very high concentrations, suggesting the possible

11 detrimental consequences of high intakes of flavanols (Manach et al., 2005; Scalbert &

Williamson, 2000).

Flavonols may be found in various food sources such as apples, strawberries, raspberries, grapes, teas, lettuce, green beans, onions, scallions, kale, and others

(D’Andrea, G., 2015). Certain flavonols have been reported to pose a wide range of pharmacological properties such as anticancer, antioxidant, cardioprotective, neuroprotective, anti-inflammatory, anti-diabetic, anti-osteoporotic, and anti-allergic properties (Caldern-Montao et al., 2011). The studies included on this paper confirmed that quercetin, dihydroquercetin, kaempferol, galangin, isorhamnetin, taxifolin, hyperoside, rutin, myricetin, dihydromyricetin, and morin modulate QR either by increasing its activity or mRNA gene expression in various cells and animal studies

(Tables 2 - 12). However, Heike et al. (2009) did not show any changes upon the treatment with quercetin on Wistar Unilever, possibly due to the high concentration prescribed to the animals. In the case of quercetin, it is assumed that its bioactivity mainly comes from it being metabolized in the intestine and/or liver into natural occurring conjugated forms that are easily absorbed and transported to other tissues

(D’Andrea, G., 2015). A few studies utilized the Caco-2 cell line model, which has been proved to be a useful model for absorption and to test the effects of toxic compounds.

(Niestroy et al., 2011). Common sources of quercetin in a Western diet include teas, red wine, fruits and vegetables and it may be available as dietary supplements in doses ranging from 200 to 1200 mg (D’Andrea, G., 2015).

Flavones can be found in teas, herbs, and vegetables such as chamomile, parsley, tansley leaf, rooibos bush tea, fenugreek seed, peppermint, oregano, shiso, rosemary,

12 sage, green tea, black tea, oolong tea, and celery (Hostetler et al., 2017). Flavones such as apigenin, diosmetin, luteolin, scutellarin, 4’dimethylnobiletin, and rutin significantly modulated QR activity and its mRNA expression (Tables 2 - 12). However, luteolin delivered to HepG-2 cells at concentrations of 3 and 10 µM decreased QR mRNA expression, given that the treatment dose was too high from the typical physiological concentration of flavonoids in the blood when compared to humans (Zhang et al., 2014).

Flavanones which can be found vastly in citrus such as sweet orange, mandarin, clementine, lemon, lime, grapefruit, bergamot, sour orange, tangelo, cedro, kumquat, and tangor pose protective effects against oxidative stress and the prevention of chronic diseases (Barreca et al., 2017). Eriodictyol, hesperetin, hesperidin, pinostrobin, naringenin, and naringenin-7-glucoside elevated QR activity and its mRNA gene expression in various cell lines and animal studies. The health promoting properties of these flavanones may be an important source of antioxidants to humans since citrus are cultivated worldwide (Barreca et al., 2017).

Isoflavones are commonly found in legumes of the family Fabaceae, red clover, white clover, and alfalfa. However, the main source of isoflavones in the human diet comes from soy and soy-derived products, which can provide about 1.5 mg/g (Křížová et al., 2019; Rizzo & Baroni, 2018). The presented studies demonstrated that aglycone and some metabolites of isoflavones modulated QR activity and mRNA gene expression in various cell lines and animal studies (Tables 2 - 12). Isoflavones have been suggested to inhibit the proliferation of cancer cells. For instance, the prevalence of breast cancer in

Asian countries where the daily intake of isoflavones has been estimated to be around 25-

50 mg, is higher compared to western countries where the daily intake of isoflavones is

13 less than 2 mg (Křížová et al., 2019; Messina et al., 2006; Van Erp-Baart et al., 2003).

Various studies demonstrated that treatment with isoflavones in vitro promoted the induction of NQO1, which plays an important role in the inhibition of oxidative stress; thus, reducing the initiation and progression of cancer cells (Bianco et al., 2005; Wang et al., 1998)

4.2 Bioavailability of Flavonoids

Generally, flavonoids will be found in food sources as glycosides, attached to one or more monosaccharides. (Williamsom, 2004). The forms in which flavonoids may be present will determine their bioavailability. Cells in the small intestine via enzymes (e.g., lactase phlorizin hydrolase) can hydrolyze these glycosides into their corresponding aglycones. Similarly, the intestinal microbiota can also hydrolyze the sugar moiety of glycosides and yield its aglycone forms that may be readily absorbed by the colon.

Intestinal microbiota may also metabolize these aglycones into low molecular weight catabolites such as phenolic or aromatic acids which may become bioavailable (Pereira-

Caro et al., 2016). Once the aglycones have been able to enter intestinal epithelial cells, the phase I (e.g., cytochrome p450) and phase II enzymes (e.g., NQO1, heme-oxygenase

1 heat shock protein 32 (HO-1), glutathione-S-transferase (GST), DP- glucuronosyltransferase 1A6, sulfotransferases and methyltransferases) produce conjugated metabolites such as monoglucuronides and sulfates. When intestinal conjugation occurs, these molecules reach the liver where phase II enzymes may continue methylation or sulphation to yield multiple conjugates or metabolites, resulting in more polar compounds that can perhaps target tissues or be excreted by the body. Thus, blood systemic circulation and urine rarely show any levels of aglycones, but instead flavonoids

14 are found as metabolites in the blood (Murota et al., 2018). The chemical structure of flavonoids is important since the clinical relevance of these compounds will be determined by their capacity to be converted into metabolites (e.g., daidzein → equol), as it has been shown that the biological activities of certain metabolites differ from their aglycone forms (Atkinson et al., 2005; Ingram et al., 1997; Murota et al., 2018; Setchell et al., 2002).

4.3 Mechanisms by Which Flavonoids Increase QR Activity and Gene Expression

It is believed that flavonoids and other phytonutrients have the capacity to modulate the induction of phase II enzymes by promoting the translocation of Nrf2 into the nucleus. During normal cellular conditions or periods of oxidative stress, the gene expression of QR is regulated by two distinct elements, which are the antioxidant response element (ARE) also known as the electrophile response element (EpRE), and the xenobiotic response element (XRE) also known as the aryl hydrocarbon receptor

(AhRE) (Nioi & Hayes, 2004). Although multiple transcription factors (e.g., Jun, Fos,

Fra, Maf, Raf) may interact with the ARE/XRE core promoter region, the Nrf2 transcription factor is believed to be the major regulator of cytoprotective responses to oxidative stress (Li et al., 2012; Ross et al., 2000). Under homeostatic conditions, a redox-sensitive E3 ubiquitin ligase substrate known as Keap1, regulates Nrf2 in the cytosol by binding two molecules of Keap1 to Nrf2 (Itoh et al., 1999; Tonelli et al.,

2018). Under conditions of oxidative stress, the reactive cysteine residues of Keap1 are oxidized, resulting in the dissociation of the Nrf2-Keap1 complex. Then, Nrf2 is released in the cytosol and translocates to the nucleus to form a heterodimer with members of the sMaf protein family (MafF, MafG, MafK) that bind in a sequence-specific manner to the

15 ARE core promoter region (Tonelli et al., 2018); thus, resulting in the gene transcription of phase II enzymes such as QR. Although the precise mechanism by which flavonoids disrupt the Nrf2-Keap1 complex is not yet understood; however, substantial evidence shows that flavonoids modulate the expression and activity of QR via Nrf2 binding to the

ARE (Figure 3).

4.4 Recommended Intakes of Flavonoids

It has been estimated that Americans consume a daily mean intake of 1 g/day of flavonoids (Kahnau, 1976; Scalbert & Williamson, 2000). Chun et al. (2007) reported the major dietary sources of flavonoids in the US come from tea, citrus fruit juices, wine, and citrus fruits. It was noted that teas are the most important source of flavonoids among

Americans, especially for flavan-3-ols and flavonols which provide 167 mg of daily flavonoid intake. However, the flavonoid density of diets in the US increases with age, income, and is higher in women, Caucasians, and vitamin supplement users. Currently in the United States, there are no dietary reference intakes (DRI) available for flavonoids or other food bioactive components, given that these molecules have not yet been established as essential nutrients. However, it is of critical importance to be able to establish recommended intakes for phytonutrients, given that substantial scientific evidence has demonstrated that certain isolated flavonoids can help to prevent the onset of chronic diseases. It is necessary to do more research on this topic through more randomized controlled clinical trials, observational studies, animal studies, and in vitro studies. It has been assumed that by establishing more DRI-like guidelines, people will be more motivated to consume more flavonoid-rich fruits and vegetables or well formulated dietary supplements (Wallace et al., 2015). Given that the public already has access to

16 dietary supplements of phytonutrients, it is critical that they understand the adverse health effects of overconsuming these products. More research on the health-promoting effects of phytonutrients (e.g., flavonoids) is necessary to understand the specific benefits of isolated phytonutrients and to provide access of this information to the public. However, a study in which the subjects consumed one medium orange resulted in serum naringenin and hesperetin concentrations up to 0.1 µM (Brett et al., 2009). Erlund et al. (2002) reported that a high vegetable diet including one glass of orange juice, one half orange, and one half mandarine provided 132 mg of hesperetin and 29 mg of naringenin. Thus, resulting in mean plasma hesperetin concentrations of 325 nM and mean plasma naringenin concentrations of 112.9 nM. Although the animal studies included in this paper did not justify how the doses may be of clinical-relevance to humans, in a study where gavage administration of naringenin to female mice at 4 mg/kg bw (body weight) resulted in plasma concentrations of 0.5 µM. However, this study discussed that a similar peak plasma concentration of 0.6 µM is also observed in man following consumption of

400 to 760 mL of orange juice. (Breinholt et al., 2004; Erlund et al., 2001).

4.4 Future Studies

Further studies need to be conducted to understand the appropriate dietary recommended intake of flavonoids in humans. More clinical trials need to be done to be able to understand how the health promoting effects of flavonoids may differ between age, sex, habitual diet, drug interactions, genotype, and gut microbiome in humans

(Cassidy & Minihane, 2017). It is important that future in vitro and in vivo studies work with concentrations of flavonoids of physiological relevance to be able to understand the mechanisms in which flavonoids affect QR activity. In addition, it is important to

17 understand how other nutrients may interact with the bioavailability of flavonoids. Future animal studies should consider using doses that may be applicable to a human diet.

Furthermore, researchers should investigate the effects of flavonoid metabolites to see if they affect QR activity or gene expression. It is also crucial to understand the levels of toxicity of these compounds since some companies already distribute dietary supplements available over the counter.

18 CHAPTER 5: Conclusion

The information provided in this article review demonstrates that different flavonoids from all its subclasses can modulate QR activity and its gene expression. Most of the studies presented utilized flavonoids in the form of aglycones, with a few exceptions including some metabolites. A few studies reported no change or decreased activity of QR and its gene expression due to a high dose provided to the cells or animals.

It is important to consider the relevance of using concentrations of flavonoids within a physiological range, while considering the adverse effects of high intakes. From the studies presented it is not possible to determine dietary recommendations of flavonoids for humans, given that more randomized clinical controlled trials need to be done.

However, this paper can serve as a guide for researchers to include more relevant tools to successfully study the health-promoting effects of flavonoids in humans. It is evident that some flavonoids are excellent free radical scavengers that have the capacity to induce the gene expression of NQO1 by promoting the translocation of Nrf2 into the ARE promoter region. The studies presented suggest that flavonoids may be powerful pharmacological agents to fight against the development and progression of chronic diseases such as cancer, cardiovascular disease, or neurodegenerative diseases.

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32 APPENDIX: Tables & Figures

Table 1 Common Dietary Flavonoids (Higdon et al., 2016)

Flavonoid Dietary Flavonoids (aglycones) Some Common Food Sources Subclass Anthocyanidins* Cyanidin, Delphinidin, Malvidin, Pelargonidin, Red, blue, and purple berries; red and Peonidin, Petunidin purple grapes; red wine Flavan-3-ols Monomers (Catechins): Teas (particularly white, green, and (+)-Catechin, (-)-Epicatechin, (-)- oolong), cocoa-based products, grapes, Epigallocatechin, (+)-Gallocatechin; and their berries, apples gallate derivatives Dimers and Polymers: Apples, berries, cocoa-based products, Proanthocyanidins# red grapes, red wine

Theaflavins, Thearubigins Black tea

Flavonols Isorhamnetin, Kaempferol, Myricetin, Quercetin Onions, scallions, kale, broccoli, apples, berries, teas

Flavones Apigenin, Luteolin, Baicalein, Chrysin Parsley, thyme, celery, hot peppers

Flavanones Eriodictyol, Hesperetin, Naringenin Citrus fruit and juices, e.g., oranges, grapefruits, lemons

Isoflavones Daidzein, Genistein, Glycitein, Biochanin A, Soybeans, soy foods, legumes Formononetin

*Anthocyanidins with one or more sugar moieties (anthocyanidin glycosides) are called anthocyanins. #Proanthocyanidin oligomers formed from (+)-catechin and (-)-epicatechin subunits are called procyanidins.

2 Summary of the effects ofanthocyanidins on NQOI gene expression and QR activity in various cell lines (T induction,! inhibition,+-> no change). Authors Tvoe of Cell Duration Treatment Concentration Results Ma et al. (20 19) HT-29 (human colorectal 24 h Anthocyanin 1, 5, 10, 50 µM Tpro tein level adenocarcinoma) Thummayot et al. SK-N-SH (human 26 h Cyanidin 20 µM Tpro tein level (2018) neuroblastoma) Shih et al. (2007) Clone-9 (rat liver) 24 h Cyanidin 50 µM Tacti vity and protein level

Li et al. (2019) IB-6-P (mouse skin 6 h, 5 d Pelargonidin 30, 50 µM TmRNA expression and eoidermal) protein level Shih et al. (2007) Clone-9 24 h Kuromanin 50 µM Tacti vity and protein level

Shih et al. (2007) Clone-9 24 h Delphinidin 50 µM Tacti vity and protein level

Shih et al. (2007) Clone-9 24 h Malvidin 50 µM Tacti vity and protein level 34 le 3

Summary of the effects offlavan-3-ols on NQO I gene expression and QR acti,~ty in various cell lines (T induction, J, inhibition,+-+ no change).

Authors Tvne of Cell Duration Treatment Concentration Results Muzolf-Panek et al. Hepa-lclc 7 24h Epigallocatechin 100 µM TmRNA expression (2008) (mouse heoatorna) gallate Warwick et al. (2012) 142Br (human primary skin 24 h Catechin 20µM TmRNA expression fibroblast) 35 le 4

Summary of the effects offlavonols on NQOl gene expression and QR activity in various cell lines (T induction, l inhibition, +-+ no change). Authors Tvoe of Cell Duration Treatment Concentration Results Warwick et al. (2012) 142Br 24 h Quercetin 5, 10, 20 µM T mRNA expression

Xu et al. (2016) ARPE-19 (human retinal 58 h Quercetin 200µM T mRNA expression oie:ment epithelial) Valerio et al. (2001) MCF-7 (human breast 24h Quercetin 15 µM f protein level carcinoma) Tanigawa et al. (2007) HepG-2 (human hepatocyte) 12 h Quercetin 5, 10, 20,40 µM T mRNA expression Niestroy et al. (2011) Caco-2 (human colon 48 h Quercetin 1, 10 µM T mRNA expression carcinoma) Weng et al. (2017) ARPE-19 24 h Quercetin 6, 12, 24, 48 uM i protein level Uda et al. (1997) Hepa-lclc 7 24 h Ouercetin 0- 100 uM i activitv Liang et aL (2013) HepG-2 48 h Dihvdroquercetin 10, 20, 30 uM jprotein level Niestroy et al. (2011) Caco-2 48 h Kaempferol 1, 10 µM T mRNA expression 36 Uda et al. (1997) Hepa-lclc 7 24 h Kaempferol 0- 100 uM i activity Uda et al. (1997) Hepa-lcl c7 24 h Galane:in 0- 100 uM i activitv Yang et al. (2014) HepG-2 12 h lsorhamnetin 30 uM i protein level Xie et al. (2017) ARPE-19 24 h Taxifolin 10, 20, 50 µM T mRNA expression and protein level Chen et al. (2017) HK-2 (human kidney 28 h Hyperoside 50,100,200 µM T mRNA expression and proximal tubular) protein level Karakurt et al. (2016) HepG-2 48 h Rutin 52.7 µM T mRNA expression and protein level Table 5

Swnmary of the effects of flavones on NQO 1 gene expression and QR activity in various cell lines (T induction, l inhibition, <-+ no change). Authors Type of Cell Duration Treatment Concentration Results Paredes-Gonzalez et al. JB6-6P 5 d Apigenin 1.56, 6.25 µM f mRNA expression and (2014) protein level Xu et al. (2016) ARPE-19 30 h Apigenin 400µM i mRNA and protein Wang C et al. (2018) L02 (human normal 24 h Diosmetin 30µM f mRNA expression and heoatocvte) orotein level Kitakaze et al. (2019) HepG-2 24 h Luteolin lOOnM i protein level Zhang et al. (2014) HepG-2 10m Luteolin 3, 10 uM l mRNA expression Tan et al. (2014) NHBE (normal human 24 h Luteolin 2.5, 5, 10, 20, 40 f protein level bronchial eoithelial) uM 37 Table 6

Summary of the effects of flavanones on NQO I gene expression and QR activity in ,·arious cell lines CTinduction , ! inhibition, .... no change).

Authors Type of Cell Duration Treatment Concentration Results Johnson et al. (2009) ARPE-19 24 h Eriodictyol 10,20,30,50, 100 Tpro tein level uM Chen et al. (2019) HK-2 1 h Hes.,..,-etin 2.5, 5, 10 11M t orotein level Fahey et al. (2002) Hepa-lclc 7 n/a Pinostrobin 0.5 uM t activity Raianna et al. (2019) MIN-6 (mouse insulinoma) 24 h Nariniz.enin 50,100 11M t orotein level Han et al. (2008) H9C2 (rat cardiac) 24h Naringenin-7-0- 10, 20, 40 µM T mRNA expression izlucoside 38 7

Summary oftb.e eff'ect3 ofisoflavones on NQOl gene expression and QR activity in various cell lines ( induction, ! inhibition,+-+ no change).

Authors Type of Cell Duration Treatment Concentration Results Y annai et al. (1998) Heoa-lclc 7 24 h Genistein 20, 30, 40 u.M: i activity W ang et al ( 1998) Colo205 (human colon 48 h Genistein 0.01 - 10.0 µM i activity and mRNA cancer) exoression Bianco et al. (2005) MDA-MB-23 1 (mouse 48 h Genistein IO~M mRNA expression and breast cancer,, protein le\·el Froy en et al. (2011) Hepa-lclc 7 1,24,48 h Genistein 1, 5, 25 µM i mlUfA expression, activity, protein le,·el

Y annai et al. (1998) Heoa-lclc 7 24h D aid.zein 20, 30, 40 lili.f i activity Zhang et al (2013) EAhy926 (human umbilical 10 h Da:idz.ein 500nM i protein level vein e-ndothe.lial) Froy en et al. (2011) Hepa-lc lc7 1,24,48 h Daidz.ein 1, 5, 25 µM i m.Ri"\fAexpre ssion, activitv, protein level

39 Froy en et al. (2011) Hepa-lclc 7 1,24,48 h Equol 1, 5, 25 µM i m.Ri"\fAexpressi on, activitv vrotein level Zhang et al (2013) EAhy926 (human umbilical 10 h S- (-) equol 250 nM i protein level vein endothelial) Bianco et al. (2005) MDA-1'.ffi.-231 48 h BiochaninA IO~M i protein level

W ang et al. ( 1998) Colo205 48 h BiochaninA 0.01 - 10.0 µM i activity and mRNA expression W ang et al ( 1998) Colo205 48 h Coumestrol 0.0 1 - 10.0 µM i activity and mRNA expression Park et al. (2011) C6 astrogloma 48 h T ectorigenin 10, 25, 50, 100 µM mRNA expression and protein le\·el Park et at (2011) C6 astrogloma 48 h Glycitein 10, 25, 50,. 100 µM mRNA expression and orotein le\·el Table 8

Summary of the effects offlavan-3-ols on NQO l gene e.-..pression and QR activity from Various Animal Studies Ci induction, ! inhibition,+-+ no change). Authors Tyne. of Cell Duration Treatment Concentration Results Wang et al (2015) Kunming 6d (-)-epigallocatechin-3- 75 mg/kgbw protein level and mRNA 1fouse Gallate iniection exnression Heike et al (2009) Wistar Unilever (rat) 22 d Catechin 2 g,kgdiet ! activity and mRNA Pxnre.ssion 40 le 9

Summary of the effects offlavonols on QOl gene expression and QR activity from Various Animal Studies CTindu ction, 1 inhibition,+-+ no change). Authors Type of Cell Duration Treatment Concentration Results Heike et al. (2009) Wistar Unilever 22d Quercetin 2 g/kg diet +-+ activity and mRNA expression Maturu et al. (2018) C57BL/6J (mice) 4d Quercetin 20 mg/kg bw Tprotein level and mRNA iniection ex.oression Wei et al. (2017) C57BL/6J 10 d Que.rcetin 80 mg/kg bw Tprotein level and mRNA exoression Sun et al. (2020) Broiler Chicken 21 d Quercetin 200 mg/kg diet Tprotein level and mRNA expression Vanhees et al. (2012) C57BL/6J 102.5 d Quercetin 302 mg/kg diet l mRNA expression Zhang et al. (2016) C57BL/6J 5w Myricetin 100 mg/kg bw TmRNA expression gavage Chu et al. (2018) Sprague Dawley (rat) 25 d Dihydromyricetin 20, 50 mg/kg bw Tprotein level and mRNA gavage expression 41 Sang et al. (2017) Sprague Dawley 8w Morin 50 mg/kg bw Tprotein level and mRNA exoression Kawabata et al. (1999) F344 (Fischer 344 Albino 10 w Morin 100,5 00 ppm Ta ctivity Rat) diet Tanaka et al. (1999) F344 4,28, 32 Morin 500 ppm diet Tac tivity w le 10

Summary of the effects of flavones on NQO I gene expression and QR activity from Various Animal Studies (l induction, ! inhibition, <-+ no change). Authors Tvn.o of Cell Duration Treatment Concentration Results Tan et al. (2020) Sprague Dawley 24 h Luteolin 10, 20 mg/kg bw Tprotein level iniection Tan et al. (2018) Wistar UnileYer 56 d Luteolin 80mg/kgbw Tprotein level 2ava2e Li ct al. (2016) C57BU6J 7d Lutcolin 20, 50 mg/kg bw Tprotein level and mRNA aavaszc cxorcssion Yang et al. (20 I 6) Kunming Mouse 24 h Luteolin I00mg/kg bw Tprotein level 2ava2e Xu et al. (2014) !CR Mice 30m Luteolin 30mg/kg bw Tprotein level and mRNA cxoression Khan et al. (20061 Swiss Webster (mice) 7d Aoiszcnin 2.5. 5 mg/kg diet t activity Yang el al. (2018) Kunming Mouse 4w Apigcnin 50 mg/kg bw Tprotein level

42 gavage Guo et al. (2019) C57BU6J 24 h Apigetrin 15, 30 mg/kg bw Tprotein level "ava"e Fan H et al. (2017) Sprague Dawley 4w Scutellarin 50,100,300 Tpro tein level m2/ka diet Wu et al. (2015) CD-I Mice 30m 4 'Dimethylnobiletin 2, 4 µmo! Tprote in level tooicallv treated Balakinaet al. (2016) Wistar Unilever 14 d Rutin 400ma/kabw i mRNA exoression Table 11

Summary of the effects offlavanones on NQOl gene expression and QR activity from Various Animal Studies (T induction, ! inhibition, <-+n o change).

T e of Cell Duration Concentration Wistar Unilever 14 d 2 1 d 50m bw 7d 50m bw 43 le 12

Summary of the effects of isoflavones on NQO 1 gene expression and QR activity from Various Animal Studies (j induction, 1 inhibition, ...... no change). Authors Tvoe of Cell Duration Treatment Concentration Results Bai et al. (2019) 129/sv Mice 4w Genistein 5 mg/kg bw Tprotein level iniection Wiegand et al. (2009) Wistar Unilever 22 d Genistein 2 g/kg diet Tactiv ity and mRNA expression Miao et al. (2018) Spra1me Dawlev 2w Genistein 10 mg/kg i mRNA expression Froven et al. (2009) Swiss Webster 7 d Genistein 1500 mg/kp-diet T activitv Appelt & Reicks (1999) Sprague Dawley 2w Genistein 0.03, 0.4, 0.81 Tactiv ity mg/g diet Jia et al. (2019) Sprague Dawley 4w Genistein 5, 25 mg/kg bw Tprotein level

44 gavmi:e Appelt & Reicks ( 1999) Sprague Dawley 2w Daidzein 0.03, 0.4, 0.81 Tacti vity mg/g diet Figure 1 Basic Structures of Flavonoids Subclasses (Higdon et al., 2016)

Allthoc)'Rllidms flavonol \ . I.

5 '1:

45 Flavones Flavanones Tuotlavones

Figure 2

The QR assay is based on the production of a blue color when MTT is reduced nonenzymatically by menadiol that is generated enzymatically from menadione by quinone reductase (Prochaska & Santamaria, 1988).

6-PHOSPHO GLUCOSE GLUCONATE 6-PHOSPHATE

NAOPH NA0P

REDUCED MTT MTT (FORMAZAN DYE)

46 Figure 3

The mechanism by which flavonoids translocate Nrl2 into the nucleus are unkno,vn; however, it is believed to occur via the disruption of the Nrl2-Keapl complex.

Flavonoids

Liver Cel I Cytosol

Nucleus