Research aims of the project
Flavonoids are dietary antioxidants assumed to protect against a number of diseases which have been linked to oxidative stress, such as coronary heart disease, neurodegenerative diseases or cancer (Knekt et al. 2002). They are therefore available on the market as high dose dietary supplements. However, for two reasons it is far from being clear that flavonoids exert only beneficial actions: 1) The hydroquinone or catechol structure of some of the most potent antioxidative flavonoids renders them prone to redox cycling and formation of reactive oxygen species within the cell. 2) Cells react to oxidative stress by a coordinated adaptive response and exogenous antioxidants may interfere with this stress response.
Our project aims at elucidating the mechanisms by which dietary flavonoids influence the induction of detoxification enzymes in the intestinal tract via activation of the antioxidant responsive element (ARE). It is an additional aim of our study to describe in a cooperation with project 1 the role of flavonoids in the cross talk between ARE signaling and aryl hydrocarbon receptor (AhR) signaling and to test in a cooperation with project 5 the influence of cadmium on ARE signaling.
State of the art of current research
The ARE is found in the promoter region of a battery of genes coding for detoxification enzymes of phase II xenobiotic metabolism such as NADPH-quinone oxidoreductases (NQO), glutathione transferases (GST) and the g-glutamyl cysteine synthetase (GCS). These enzymes protect the cell against prooxidant stress. Ironically, the denomination of the ARE was derived from the observation that it can be activated by phenolic antioxidants although it was later found that their prooxidant metabolites actually give rise to the activation. While structure-activity relationship studies on ARE activation have early been performed with synthetic polyphenols only sporadic observations have been published on ARE activation by flavonoids including (-)-epigallocatechin gallate (Chen et al. 2000), quercetin (Valerio et al. 2001) and fisetin (Hou et al. 2001). However, two studies have been published which show that dependent on their chemical structure some flavonoids can induce the quinone reductase indicating that they may act through the activation of the ARE of the NQO1 promoter (Uda et al. 1997, Yannai et al. 1998).
The transactivating factor of the ARE has been identified to be a member of the Nrf (NF-E2-related factors) family of basic leucine zipper transcription factors, Nrf2 (Nguyen et al. 2000). Nrf2 is activated by release from binding to the cytosolic protein Keap1 (Itoh et al. 1999) which bridges Nrf2 to the Cul3-based E3 ligase regulating Nrf2 ubiquitylation and thus targets Nrf2 for proteasome-mediated degradation (Cullinan et al. 2004). Treatment with the synthetic phenolic antioxidant tert-butyl hydroquinone (TBHQ) which serves as a model activator of the ARE leads to nuclear accumulation of Nrf2. This is not due to increased gene expression of Nrf2 (Dhakshinamoorthy et al. 2005) but to Nrf2 stabilization paralleled by increased Keap1 ubiquitylation (Li et al. 2005). Stabilisation of the Nrf2 protein was also reported for the toxic heavy metal ion cadmium (Steward et al. 2003). It is at present not clear whether Keap1 sequesters Nrf2 in the cytoplasm by a Crm1/exportin-dependent nuclear export mechanismsm (Velichkova and Hasson (2005) or whether Keap1 is able to transiently shuttle into the nucleus to promote the ubiquitylination of Nrf2 activity (Nguyen et al. 2005).
A link between phase I and phase II metabolism is provided by the fact that a xenobiotic responsive element (XRE) is present in the Nrf2 promoter indicating that Nrf2 transcription is regulated by the AhR (Nioi and Hayes 2004, Miao et al. 2005).
There is no indication in the literature whether flavonoids share an attack with TBHQ in the processes outlined above. There are a few papers on the influence of plant-derived ARE activators such as resveratrol (Chen et al. 2005) and chlorogenic acid (Feng et al. 2005) on the activity of upstream kinases in Nrf 2 signaling. Flavonoids inhibit a variety of kinases. It has been assumed that they compete with the binding of ATP (Matter et al. 1992). In a comparative study with a series of flavonoids on PI3 kinase, EGF receptor tyrosine kinase and PKC it has been shown that there exists a selectivity of single compounds for single kinases (such as myricetin towards PI3 kinase or genistein towards receptor tyrosine kinase) but there is no absolute specificity (Huang et al. 1999). An activation of the three downstream acting MAP kinases ERK, JNK-1 and p38 by (-)-epicatechin gallate and quercetin has been shown (Chen et al. 2000, Sidhu et al. 2001).
Participant´s previous work in the field
The laboratory of RK has been involved in the identification of prooxidant mechanisms of the synthetic phenolic food antioxidant butylhydroxyanisole (BHA) and its demethylated metabolite TBHQ, the model activator of the ARE. We have shown that TBHQ autoxidizes to the TBQ· semiquinone radical and that TBQ· then enters a redox cycle producing superoxide anion radicals and subsequently H2O2 and hydroxyl radicals (Kahl et al. 1989, Bergmann et al. 1992). Our findings have provided an explanation for the mechanism of ARE activation by BHA and for the way by which BHA and TBHQ induce detoxicating phase II enzymes.
In the last few years, RK and WW have performed a comparative study of a number of flavonoids with respect to their cellular uptake, their protective actions against oxidative stress and their prooxidative and proapoptotic actions [RK 1 – 5, 10, 13; WW 1 – 5, 7]. We have shown that the cellular uptake of flavonoids is closely related to distinct structural elements. The flavonoids that were taken up by the cells were metabolized mainly to glucuronides. We found protective effects of flavonoids in low concentrations against oxidative stress, but in higher concentrations also adverse effects were detected (induction of DNA strand breaks, oxidative stress and apoptosis). WW performed several studies on mechanistic effect of cadmium toxicity (e.g. induction of oxidative stress and induction of apoptosis) [WW 6, 10-14].
Title of thesis- “Role of dietary flavonoids in the activation of the antioxidant responsive element and the induction of phase II drug metabolizing enzymes”
In primary human IEC, ARE–driven transcription, subcellular Nrf2 and Keap1 localization, Nrf2 stabilization and activation of upstream kinases will be studied. In mice in vivo, expression of the NQO1 gene will be measured in intestinal and liver tissue preparations and gel shift experiments will be performed.