Dietary flavonoids and their colonic catabolites as cytoprotective antioxidants against oxidative stress March, 2019 SAYAKA NAKASHIMA Graduate School of Environmental and Life Science (Doctor Course) OKAYAMA UNIVERSITY, JAPAN CONTENTS CONTENTS 2 LIST OF TABLES AND FIGURES 6 ABBREVIATION 7 ABSTRACT 9 CHAPTER 1 General Introduction 12 1.1 Functional foods 12 1.2 Flavonoids 12 1.2.1 Chemical structure and classification of flavonoids 13 1.2.2 Dietary sources of flavonoids 13 1.2.3 Metabolism and absorption of flavonoids 14 1.2.4 Biological activities and health benefits of flavonoids 15 1.3 Oxidative stress and disease 16 1.3.1 Reactive oxygen species 16 1.4 Cytoprotective system against xenobiotics and oxidative stress 17 1.4.1 Aryl hydrocarbon receptor signaling pathway 18 1.4.2 Keap1-Nrf2 signaling pathway 18 1.4.3 Interaction between Keap1/Nrf2 pathway and AhR pathway 19 1.5 Click chemistry 22 1.5.1 The Copper (I) -catalyzed azide alkyne cycloaddition 22 1.6 Study outline 23 2 CHAPTER 2 3,4-Dihydroxyphenylacetic acid, a catabolite of dietary quercetin glycosides 24 produced by gut microbiota have cytoprotective effects against oxidative stress 2.1 Introduction 24 2.2 Materials and methods 27 2.2.1 Materials 27 2.2.2 Cell culture and treatment 27 2.2.3 DPPH radical scavenging assay 27 2.2.4 Superoxide scavenging assay in the xanthine/xanthine oxidase 28 system 2.2.5 Determination of hydrogen peroxide formation 28 2.2.6 Reverse transcription-polymerase chain reaction (RT-PCR) 28 2.2.7 Glutathione S-transferase (GST) activity assay 29 2.2.8 Glutathione titration 29 2.2.9 Cell viability determination with MTT assay 30 2.2.10 Statistical analysis 30 2.3 Results 31 2.3.1 DOPAC has radical scavenging and prooxidative activities. 31 2.3.2 DOPAC induces phase 2 drug-metabolizing enzymes in 33 Hepa1c1c7 cells. 2.3.3 DOAPC increases glutathione S-transferase activity in RL34 36 cells and cellular glutathione content in Hepa1c1c7 cells. 2.3.4 DOPAC protects Hepa1c1c7 cells against hydrogen peroxide 38 induced oxidative stress. 2.4 Discussion 40 3 CHAPTER 3 3,4- Dihydroxyphenylacetic acid has cytoprotective effects against oxidative 44 stress through direct modification of Keap1 and aryl hydrocarbon receptor 3.1 Introduction 44 3.2 Materials and methods 47 3.2.1 Materials 47 3.2.2 Instrumental methods of chemical analysis 47 3.2.3 Cell culture and treatment 47 3.2.4 Synthesis of DOPAC propargyl ester 48 3.2.5 CuAAC reaction with DPE and benzyl azide 48 3.2.6 RNA extraction and RT-PCR 49 3.2.7 Competitive inhibition of DPE-modification in cells and in cell 50 lysate 3.2.8 Pulldown assay and western blotting 50 3.2.9 Statistical analysis 50 3.3 Results 51 3.3.1 Design and synthesis of click chemistry probe. 51 3.3.2 Evaluation of chemical and biological properties of DPE. 52 3.3.3 Detection of DPE-modified model proteins in vitro. 54 3.3.4 Detection of the intracellular DPE-modified proteins. 56 3.3.5 Identification of the target proteins of DOPAC. 58 3.4 Discussion 59 CHAPTER 4 Aureusidin, an aurone-type flavonoid with an unique structure, as a potential 61 cytoprotective agent against oxidative stress 4.1 Introduction 61 4 4.2 Materials and methods 63 4.2.1 Materials 63 4.2.2 Cell culture and treatment 63 4.2.3 Synthesis of aureusidin (AU) 63 4.2.4 RNA extraction and RT-PCR 65 4.2.5 Cell viability determination with MTT assay 65 4.2.6 Statistical analysis 65 4.3 Results 66 4.3.1 Synthesis of AU by four-step synthesis. 66 4.3.2 Cytoprotective effects of AU against hydrogen peroxide-induced 68 oxidative stress. 4.3.3 Modulating effects of AU on phase 1 and 2 gene expression. 69 4.4 Discussion 71 CONCLUSION 73 ACKNOWLEDGEMENTS 75 REFERENCE 76 5 LIST OF TABLES AND FIGURES Figure 1.1 Basic structures of flavonoid subclasses. 14 Figure 1.2 Schematic representation of drug detoxification pathway. 17 Figure 1.3 Schematic representation of Keap1/Nrf2 signaling pathway. 20 Table 1.1 Phase 2 antioxidative enzymes and their functions. 21 Figure 1.4 The Copper (I) -catalyzed azide alkyne cycloaddition. 22 Figure 2.1 Schematic representation of quercetin glycoside metabolism and absorption in the 26 large intestine. Figure 2.2 Chemical structures of phenolic catabolites of quercetin glycosides. 31 Figure 2.3 Radical scavenging and prooxidative potentials of the phenolic acid catabolites. 32 Figure 2.4 Modulating effects of the phenolic acid catabolites on the gene expression of the 34 drug metabolizing enzymes. Figure 2.5 Concentration-dependent effects of the DOPAC on the gene expression of the drug 35 metabolizing enzymes. Figure 2.6 Modulating effects of the DOPAC and quercetin on the total GST activity and the 37 intracellular GSH level. Figure 2.7 Effect of DOPAC on the hydrogen peroxide-induced cytotoxicity in Hepa1c1c7 39 cells. Figure 3.1 DOPAC modifies a protein sulfhydryl group in a phenol oxidase-dependent 45 manner. Figure 3.2 The strategy for detecting DOPAC-targeting proteins using DPE as a click 51 chemistry probe. Figure 3.3 Esterification of DOPAC with 2-propyn-1-ol to give DPE. 52 Figure 3.4 Chemical and biological properties of DPE and DOPAC. 53 Figure 3.5 CuAAC reaction of DPE with an azide-linked tag molecule. 55 Figure 3.6 Detection of the intracellular DPE-modified proteins. 57 Figure 3.7 Detection of the DPE-modified Keap1 and AhR by pulldown assay. 58 Figure 4.1 Chemical structure of aurone and aureusidin. 62 Figure 4.2 Synthesis of AU in four steps from phloroglucinol. 67 Figure 4.3 Cytoprotective effects of AU on hydrogen peroxide-induced toxicity. 68 Figure 4.4 Modulating effects of AU on HO-1 and NQO1 gene expression. 69 Figure 4.5 Modulating effects of AU on CYP1A1 gene expression. 70 6 ABBREVIATION AhR, aryl hydrocarbon receptor ARNT, aryl hydrocarbon receptor nuclear translocator AU, aureusidin BNPP, bis(4-nitrophenyl)phosphate CDNB, 1-chloro-2,4-dinitrobenzene CuAAC, copper(I)-catalyzed azide alkyne cycloaddition CYP1A1, cytochrome P450 1A1 DBE, DOPAC butanol ester DMSO, dimethyl sulfoxide DOPAC, 3,4-dihydroxyphenylacetic acid DPE, DOPAC propargyl ester DPPH, diphenyl-2-picrylhydrazyl DTNB, 5,5′- dithiobis (2-nitrobenzoic acid) FOX, ferrous ion oxidation-xylenol orange GAPDH, glyceraldehyde-3-phosphate dehydrogenase GCLC, glutamylcysteine ligase, catalytic subunit GSH, glutathione GST, glutathione S-transferase HO-1, heme oxigenase-1 HPA, hippuric acid HRP, horseradish peroxidase NBT, nitroblue tetrazolium NQO1, NAD(P)H:quinone oxidoreductase 1 7 OPAC, 3-hydroxyphenylacetic acid PBS, phosphate buffered saline PCA, protocatechuic acid PTSA, p-toluensulfonic acid monohydrate Q3G, 3-O-β-glucoside Q4′G, 4′-O-β-glucoside ROS, reactive oxygen species RT-PCR, reverse transcription-polymerase chain reaction SOD, Superoxide dismutase TBTA, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine TLC, thin-layer chromatography XA, xanthine xCT, cystine/glutamate antiporter XOD, XA oxidase XRE, xenobiotic response element α-MEM, α-minimum essential medium 8 Abstract Flavonoids, widely distributed secondary catabolites in plants, have attracted much attention for their preventive effects against the oxidative stress-related diseases, including cardiovascular disease, cancer and neurodegenerative disease. Flavonoids have potent free radical scavenging activities due to their hydroxyl groups attached to rings. However, recent studies have reported that bioavailability of dietary flavonoids are very low as most flavonoids usually occur in the plant-based foods and drinks as glycoside forms, and thus they are converted into conjugated metabolites or ring fission catabolites before or after entering circulation. The circulating concentration of free flavonoid aglycons is considerably lower than that of other antioxidants such us glutathione, vitamin C and E, and carotenoids, suggesting that free radical scavenging activities of flavonoids per se do not completely account for their health benefits. Therefore, it has been considered that the in vivo biological actions of flavonoids are mainly mediated by their regulation of signaling pathways via protein modification. Also, the absorption and metabolism of flavonoids should be taken into account to evaluate their effectiveness. In this study, I investigated the cytoprotective effects of the microbiota-derived catabolites of quercetin glycosides as well as aurone on the oxidative stress-induced cytotoxicity and their underlying molecular mechanisms. In chapter 2, I compared direct and indirect antioxidative activities of major phenolic catabolites including 3,4-dihydroxyphenylacetic acid (DOPAC), 3-hydroxyphenylacetic acid (OPAC), 3,4-dihydroxybenzoic acid (protocatechuic acid, PCA) and hippuric acid (HPA), which are produced from quercetin glycosides by gut microbiota. Both catechol moiety containing compounds, DOPAC and PCA exhibited DPPH radical scavenging and superoxide dismutase-like activities, whereas OPAC and HPA did not. DOPAC also enhanced the gene expressions of several phase 2 drug-metabolizing enzymes more potently than the other phenolic acid catabolites. DOPAC significantly inhibited the hydrogen peroxide-induced cytotoxicity in mouse hepatoma Hepa1c1c7 9 cells with the enhancement of the total glutathione S-transferase activity. These results suggested that DOPAC is partly responsible for the health promoting effects of dietary quercetin. In chapter 3, target proteins of DOPAC were investigated to elucidate the mechanism where DOPAC induces the phase 2 drag-metabolizing enzyme expression. I synthesized DOPAC propargyl ester (DPE) by fisher esterification of DOPAC and 2-propyn-1-ol as a novel tag-free DOPAC probe which is designed to detect the targeted proteins using the copper(I)-catalyzed azide alkyne cycloaddition (CuAAC), a representative click reaction. Various cellar proteins labeled with DPE were detected by the combination of the azide-labeled biotin and horseradish peroxidase (HRP)-streptavidin.