Along the mammalian digestive tract, which represents the internal barrier between the environment and the organism, the gastrointestinal mucosa serves as a highly selective barrier designed to permit the absorption of nutrients from the gut lumen into the circulation and to restrict the passage of potentially toxic xenobiotics (Farhadi et al., 2003). Two major routes exist for the permeation of this barrier, namely transcellular and paracellular transport, which require the respective cellular systems. Paracellular transport is mainly controlled by tight junctions (Hollander, 1992). Additionally, the cytoskeleton plays an important role in paracellular transport, beside its critical function together with adherens junctions in the maintenance of the intestinal barrier function (Alvila, 1987). For transcellular transport membrane transport proteins play a pivotal role (Baumgart and Dignass, 2002).
The small intestine principally serves as the site for absorption of nutrients, water, and xenobiotics. Accordingly, it has become apparent that enterocytes lining the intestinal mucosa are equipped with a broad range of metabolic systems such as phase I and II drug metabolising enzymes associated with different efflux pumps (Kaminsky and Fasco, 1992; Suzuki and Sugiyama, 2000). Morphologic features of the small intestine increase its metabolic competence as well as its potential for first-pass metabolism. These include the remarkable length of 7 m in humans divided into the duodenum, jejunum, and ileum (proximally to distally), the appearance of the metabolic competent epithelium as an enterocyte monolayer, as well as further amplification of the epithelial surface by villi and crypts (Kaminsky and Zhang, 2003). However, the small intestine determines the bioavailability of the majority of orally ingested drugs, beside their physiochemical properties, by metabolism and active extrusion after absorption (Suzuki and Sugiyama, 2000).
The importance of the isoenzyme cytochrome P450 (CYP) 3A4, the major phase I drug metabolising enzyme in humans, and P-glycoprotein (P-gp), the multidrug efflux pump, in limiting oral drug delivery has been suggested due to broad overlapping substrate specifities and poor oral bioavailability of joint substrates of both proteins (Wacher et al., 1996). Additionally, many compounds induce or inhibit both proteins simultaneously (Schuetz et al., 1996). Phase II drug-metabolising enzymes are able to conjugate xenobiotics with small organic donors, e.g. glutathione, by taking advantage of electrophilic functional groups already present on the molecule, or ones introduced by CYPs (McCarver and Hines, 2001). In most cases these conjugations result in detoxification and pharmacological inactivation and may render the xenobiotics to substrates for specific transport enzymes like the multidrug-resistance associated proteins (MRPs), thus facilitating excretion to the lumen.The steroid and xenobiotic receptor (SXR), or pregnane X receptor (PXR), an orphan nuclear receptor with high abundance in liver and intestine, has been shown to exert a central role in the expression of P-gp and CYP3A4 and other ABC transporters and CYP isoforms (Lehmann et al., 1998; Synold et al., 2001).
After activation by a diverse array of substances including rifampicin and hyperforin, PXR forms heterodimers with retinoic X receptor, another nuclear receptor, and then specifically interacts with a DNA sequence, the hormone-binding element. As many of the compounds that induce CYP3A4 and/or P-gp activate or bind directly to PXR, this receptor could be exploited for a screening of drug candidates, which fail to activate or inhibit this pathway (Lehmann et al., 1998; Geick et al., 2001). In addition, the constitutive androstane receptor (CAR) with high expression in the liver and intestine plays an important role in the expression of ABC transporters (Kullak-Ublick and Becker, 2003). Moreover, expression of P-gp may be induced by stress signals including heat shock, genotoxic stress, or cytokines, as respective transcription binding sites have been located in the MDR1 promotor region (Sukhai and Piquette-Miller, 2000). In the CYP1A enzyme family, each member is inducible via the aryl hydrocarbon receptor (AhR). Ligand binding to AhR induces conformational changes, which enables AhR to translocate to the nucleus, where it dimerizes with the AhR nuclear translocator protein (ARNT). These heterodimers function as transcriptional activators by binding to consensus sequences called dioxin-response elements (DRE) (Denison and Whitlock, 1995).
The general importance of drug metabolising enzymes and transport proteins is given by their control of metabolism, absorption, distribution, and excretion of endogenous substances and exogenous xenobiotics in the organism. The high abundance of these metabolising enzymes and transport proteins in the liver and in the small intestine has raised the question about the contribution of each of these organs to the first-pass metabolism of xenobiotics. The greater metabolic capacity due to a higher overall weight relative to the small intestine and a higher concentration of CYPs and of microsomal protein content, as well as the potential of absorbed systemic xenobiotics to undergo countercurrent exchange, would favour the liver (Lin et al., 1999). Nevertheless, this does not detract from the capability of the small intestine, as the first site of exposure, to metabolise or extrude orally ingested xenobiotics prior to systemic uptake. Thus, it is worthwhile to assess possible interactions of drugs of dietary origin, e.g. green tea, in view of a possible therapeutic potential and/or possible drug-drug interactions leading to reduced therapeutical effectiveness.
contents
Acknowledgements
Abbreviations
Table of contents
Summary
Aim of the thesis
1. Introduction
- 1.1 Drug absorption and metabolism in the gastrointestinal tract
1.2 ABC transport proteins
- 1.2.1 MDR1 (ABCB1)
1.2.2 MRP2 (ABCC2)
1.3 Cytochrome P450 (CYP) enzymes
- 1.3.1 CYP1A
1.3.2 CYP3A
1.4 The gastrointestinal tract as a site of immunological activity
- 1.4.1 Chemokines
1.4.2 IL-8
1.5 Green tea (Camellia sinensis (L.) O. K UNTZE , fam. theaceae)
1.5.1 General aspects
1.5.2 Pharmacological activities
2. Effect of green tea extract on reactive oxygen species (ROS) concentration under cell culture conditions
- 2.1 Introduction
2.2 Materials and methods
2.3 Results and discussion
3. Inhibitory activity of a green tea extract and some of its constituents on multidrug resistance-associated protein 2 functionality
- 3.1 Abstract
3.2 Introduction
3.3 Materials and methods
3.4 Results
3.5 Discussion
4. Induction of CYP1A by green tea extract in human intestinal cell lines
- 4.1 Abstract
4.2 Introduction
4.3 Materials and methods
4.4 Results
4.5 Discussion
5. Effect of green tea extract on tightness of intestinal epithelia
- 5.1 Introduction
5.2 Materials and methods
5.3 Results
5.4 Discussion
6. Side project: Influence of green tea extract on intestinal cytokine expression and secretion
- 6.1 Green tea extract or its constituent (-)-epigallocatechin gallate induce interleukin-8 (IL-8)mRNA and protein expression but specifically inhibit IL-8 secretion in Caco-2 cells
- 6.1.1 Abstract
6.1.2 Introduction
6.1.3 Results and discussion
6.1.4 Experimental section
6.2 Evaluation of the effect of green tea extract on intracellular IL-8 protein stability
- 6.2.1 Introduction
6.2.2 Materials and methods
6.2.3 Results and discussion
6.3 Evaluation of the influence of green tea extract on the mRNA expression of different proinflammatory mediators
- 6.3.1 Introduction
6.3.2 Materials and methods
6.3.3 Results and discussion
7. Conclusions and outlook
8. References
Curriculum vitae
