Mechanisms of toxic action of the flavonoid quercetin and its phase II metabolites Hester van der Woude Promotor: Prof. Dr. Ir. I.M.C.M. Rietjens Hoogleraar in de Toxicologie Wageningen Universiteit Co-promotor: Dr. G.M. Alink Universitair Hoofddocent, Sectie Toxicologie Wageningen Universiteit. Promotiecommissie: Prof. Dr. A. Bast Universiteit Maastricht Dr. Ir. P.C.H. Hollman RIKILT Instituut voor Voedselveiligheid, Wageningen Prof. Dr. Ir. F.J. Kok Wageningen Universiteit Prof. Dr. T. Walle Medical University of South Carolina, Charleston, SC, USA Dit onderzoek is uitgevoerd binnen de onderzoekschool VLAG Mechanisms of toxic action of the flavonoid quercetin and its phase II metabolites Hester van der Woude Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. Dr. M.J. Kropff, in het openbaar te verdedigen op vrijdag 7 april 2006 des namiddags te half twee in de Aula Title Mechanisms of toxic action of the flavonoid quercetin and its phase II metabolites Author Hester van der Woude Thesis Wageningen University, Wageningen, the Netherlands (2006) with abstract, with references, with summary in Dutch. ISBN 90-8504-349-2 Abstract During and after absorption in the intestine, quercetin is extensively metabolised by the phase II biotransformation system. Because the biological activity of flavonoids is dependent on the number and position of free hydroxyl groups, a first objective of this thesis was to investigate the consequences of phase II metabolism of quercetin for its biological activity. For this purpose, a set of analysis methods comprising HPLC-DAD, LC-MS and 1H NMR proved to be a useful tool in the identification of the phase II metabolite pattern of quercetin in various biological systems. These studies showed that the 3’- and 4’-hydroxyl groups of quercetin, (catechol hydroxyl groups) were important targets for methylation, sulfation and glucuronidation. Methylation of a catechol hydroxyl group of quercetin proved to decrease the pH-dependent radical scavenging capacity of the compound, both by increasing its pKa for deprotonation and by decreasing its electron-donating properties. Methylation of a catechol hydroxyl group had a similar effect as replacement of the hydroxyl group by a hydrogen atom. Regarding the pro-oxidant properties of quercetin, methylation of a catechol hydroxyl group of quercetin did not eliminate the pro-oxidant chemistry of quercetin, reflected in the formation of covalent adducts with glutathione upon oxidation of quercetin by horseradish peroxidase. However, methylated quercetin proved to form only 42% of the level of DNA adducts in exposed cells as compared to a similar amount of unconjugated quercetin, indicating that methylation of quercetin attenuates also this biological reactivity towards DNA. A second objective of this thesis was to obtain more insight into the possible toxic effects of quercetin by studying various mechanisms that might be relevant in the context of carcinogenesis. Quercetin appeared to have a biphasic effect on the proliferation of cancer cell lines expressing the estrogen receptor (ER). The stimulation of cancer cell proliferation was ER-dependent and appeared to occur at concentrations that are physiologically relevant in humans. With respect to the pro-oxidant activity of quercetin, peroxidase- and tyrosinase-type oxidative enzyme activity did not play a major role in the intracellular formation of covalent adducts of quercetin with DNA and protein, indicating that the formation of covalent adducts of quercetin with cellular macromolecules might also be relevant in cell types lacking oxidative enzyme activity. Furthermore, the covalent quercetin DNA adducts were of transient nature, which may either eliminate or attenuate the adverse effects of covalent DNA adduct formation. The studies presented in this thesis provided indications for the dualistic character of quercetin, regarding its role in the process of cancer development. Table of Contents page Chapter 1. General introduction, objectives and outline 9 Chapter 2. Identification of 14 quercetin phase II mono- and mixed 43 conjugates and their formation by rat and human phase II in vitro model systems Chapter 3. The effect of catechol O-methylation on radical scavenging 77 characteristics of quercetin and luteolin - A mechanistic insight Chapter 4. Formation of transient covalent protein and DNA adducts 95 by quercetin in cells with and without oxidative enzyme activity Chapter 5. Consequences of quercetin methylation for its covalent 121 glutathione and DNA adduct formation Chapter 6. Biphasic modulation of cell proliferation by quercetin at 143 concentrations physiologically relevant in humans Chapter 7. The stimulation of cell proliferation by quercetin is 157 mediated by the estrogen receptor Chapter 8. The definition of hormesis and its implications for in vitro 177 to in vivo extrapolation and risk assessment Chapter 9. Summary and future perspectives 187 List of abbreviations 203 Samenvatting 207 Dankwoord 221 Curriculum Vitae, List of publications and Training and supervision 223 plan 8 1 General introduction, objectives and outline 9 Chapter 1 1.1 From healing red peppers to polyphenol research Since the 18th century, the potency of a nutrient, later called vitamin C, to cure scurvy has been known, and the Hungarian scientist Albert von Szent-Györgyi was the first to identify the chemical structure of the compound by demonstrating its similarity to hexuronic acid (1, 2). He later re-baptized this compound ascorbic acid. The Hungarian town Szeged, where he worked at that time, was the centre of the red pepper industries. In 1934, Szent- Györgyi demonstrated this vegetable to be a rich source of ascorbic acid by developing a method to isolate the crystalline ascorbic acid from this source (3). In these days, Szent-Györgyi was asked by a colleague, who suffered from a serious haemorrhagic diathesis (increased permeability or fragility of the capillary wall), for a large amount of pure ascorbic acid to cure the disease. However, the crystalline substance was not available in sufficient amounts and therefore, Szent-Györgyi sent him red peppers to eat. As by miracle, his colleague was cured. Later on, the scientist tried to obtain the same therapeutic effect as observed for whole red peppers by using pure ascorbic acid, but he was unsuccessful. This convinced him that red peppers had to contain other substances that contributed to their therapeutic effects (4). His earlier studies on cell respiration had provided Szent-Györgyi with the insight that ascorbic acid protected plant tissues from oxidative damage resulting from a reversible interaction with a peroxidase-like enzyme. He demonstrated that a third substance, having a polyphenol structure, played an important role in this protective effect. His idea was that the peroxidase oxidized the polyphenol to a quinone metabolite, which subsequently oxidized ascorbic acid by taking up both its hydrogen atoms (5). He showed that this polyphenol belonged to a large group of yellow phenol-benzol-γ-pyran plant dyes (including flavones, flavonols and flavanones). It became clear that the members of this group of plant dyes possessed great biological activity. Based on his experience that the therapeutic effects of red peppers were larger than the therapeutic effects of a corresponding amount of pure ascorbic acid, he proposed that, possibly by interactions with ascorbic acid, these polyphenol-like dyes might also repair damaged mammalian tissues by influencing the permeability and resistance of capillary blood vessels. He was of the opinion that these substances represented a promising group of compounds with therapeutic potential against a variety of diseases. Because his experiments showed that certain members of this group of plant dyes possessed vitamin-like properties, he called the polyphenols vitamin P (4). In 1937, the importance of these findings was officially recognized when he was awarded Nobel Prize of Medicine for this pioneering work on vitamin C and vitamin P. 10 General introduction “I regret that I must conclude with many questions asked and none answered, but I hope to leave the reader with the impression that flavonoids represent one of the most exciting, broad, and hopeful fields of biological inquiry and I am glad to close on such an optimistic note”. These are the words the Nobel-Prize winning scientist closed a lecture with in 1955 (6). And he was right, because his findings on the biological activity of polyphenols were the start of decades of research on polyphenols. 1.2 Flavonoids and quercetin Polyphenols have a multitude of biological functions in the plant, including signalling, fertility, and protection against UV-light and phytopathogens (7). Polyphenols show a great diversity of structures, ranging from rather simple molecules (monomers and oligomers) to polymers (8). They can be divided into four major classes, according to the nature of their carbon skeleton, i.e. phenolic acids, flavonoids, stilbenes and lignans. O O O OH O flavone flavonol O flavanone O O + O O OH OH O anthocyanidin isoflavone flavanol (catechin) Figure 1 Structures of the major classes of flavonoids. Flavonoids (Figure 1) are the most abundant polyphenols in our diet (9). In the plant, they are products from the shikimic acid pathway (10). They consist of a three-ring structure and can be divided into several classes, depending on the degree of oxidation of the heterocyclic ring (also