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 In Nutrition and Food Science By 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