FOR RISK ASSESSMENT FEDERAL INSTITUTE. Können Pflanzeninhaltsstoffe den biochemischen. Schutz des menschlichen Darmes gegenüber

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1 H 2 N ABC CH BCRP: Breast cancer resistance protein FEDERAL INSTITUTE FR RISK ASSESSMENT Können Pflanzeninhaltsstoffe den biochemischen Schutz des menschlichen Darmes gegenüber Lebensmittelkontaminanten verstärken? Alfonso Lampen, Stefanie Hessel

2 Resorption, biotransformation and elimination of B[a]P from enterocytes Export to lumen Resorption apical membrane ABC-Transporter ABC-Transporter: p-glycoprotein MRP2 (Multidrug Resistance Protein) BCRP xidation CYP s Xenobiotica 1A1,1B1 Conjugation Transformation product transferases PAPS GSH UDPGlcUA Conjugate? blood H 2 N ABC BCRP CH Page 2

3 Expression von BCRP BCRP Tissue stomach, duodenum, colon liver, kidney brain placenta Function reduction of resorption of xenobiotica, drugs hepato-biliar excretion, renal excretion protection against xenobiotica? protection of fetus mammary gland??? Page 3

4 Metabolisierung des Lebensmittelkontaminanten Benzo[a]pyren; eine Modellsubstanz mit hohem karzinogenen Potential Benzo(a)pyren 1. Lipophiler Fremdstoff: freie Diffusion durch Membranen CYPs H 2. Hydroxylierung von B[a]P: Bildung potentieller Substrate für Phase II Enzyme; Bioaktivierung zu mutagenen Derivaten Phase I: CYPs Phase II-Enzyme -R 3. Hydrophiler Phase II- Metabolit: aktiver Transport aus der Zelle notwendig Phase II: SULT ABC Page 4

5 Transepithelial efflux of B[a]P-1-sulfate and B[a]P-3-sulfate in human intestinal Caco-2 cells [pmol/cm²] unbeh.; apikal unbeh.; basal B[a]P-1-Sulfat [pmol/cm²] unbeh.; apikal unbeh.; basal B[a]P-3-Sulfat time [h] time [h] Caco-2 cells Transwell Page 5

6 BCRP is responsible for the transepithelial transport of B[a]P-sulfates in Caco-2 cells Ko 143 Inhibition of the efflux of BAP-3-sulfate in Caco-2 cells by Ko control, apical 1200 control, basal CH 3 N H N NH [pmol/ cm²] Ko 143 (5µM), apical Ko 143 (5µM), basal - Inhibitor + Inhibitor time [h] substrate: B[a]P-3-H Page 6

7 Ah-Rezeptor Aktivierung durch B[a]P führt zu einer Induktion von CYP1A1/1B1 Kernrezeptor: Arylhydrocarbon-Rezeptor AhR Fremdstoff (z.b. PAK) Ah-Rezeptor Zellkern AhR/ARNT/Fremdstoff-Komplex ARNT DNA (DRE) ARNT BCRP? Enzyme CYP1A1 Enzyme CYP1B1 mrna Gentranskription (DRE = Dioxin response element, Promoter) Page 7

8 The role of AhR: expression of BCRP-protein in MCF-7 cells WT AhR 200 BCRP B[k]F AhR 200 : AhR-deficient (Yeh et al., 1999): small basal expression of BCRP WT AhR 200 BCRP K DBM, 50 µm Quercetin, 25 µm TBHQ, 50 µm PCB 77, 1 µm DBM = Dibenzoylmethan TBHQ= tert-butyl-hydrochinon Page 8

9 Induction of BCRP by AhR-agonists in Caco-2 cells BCRP-Proteinlevel (x-fache Erhöhung) BCRP 3 2,5 2 1,5 1 0,5 0 Kontrolle (0,1% DMS) 2,44 2,30 2,17 1,75 1 TCDD 50 nm DIM 25 µm BAP 10 µm BkF 5 µm K TCDD DIM BAP B[k]F Cl Cl TCDD N H BkF BaP N H Cl Cl DIM = 3,3`-Diindolylmethan Page 9

10 Induction of BCRP expression by phytochemicals in Caco-2 cells BCRP-Proteinlevel (x-fache Erhöhung) 3 2,5 2 1,5 1 0,5 1 2,46 2,34 1,84 1,69 1,67 1,74 2,19 H H H H Quercetin H 0 Kontrolle (0,1% DMS) TCDD 50 nm Quercetin 25 µm Curcumin 25 µm Sempervirin 25 µm Juglon 10 µm DMB 25 µm DBM 50 µm H 3 C CH 3 H Curcumin H Behandlung (3 Tage) 75 kda M K TCDD H Juglon Dibenzoylmethan Page 10

11 Effect of DBM and Quercetin on the transport of B[a]P-sulfate in Caco-2 cells Apically transported BP3S [% of total amount] * * control, apical DBM (50 µm), apical Quercetin (25 µm), apical 34 * * * 49 * Time [h] DBM = Dibenzoylmethan (aus Süßholzpflanze) Page 11

12 Conclusions There is a functional interplay between metabolism of CYP1A1/CYP1B1 substrates (PAHs), Phase II and the transport of metabolites by BCRP The ABC-transporter BCRP is responsible for the transport of B[a]P-sulfates und B[a]P-glucuronides AhR Agonists induce the gene- and protein expression of the responsible CYPs and BCRP. Regulation of BCRP-transporter is most likely AhR dependent. Phytochemicals that are AhR-agonist do have an impact on gene/protein expression of BCRP and on its functonal transport of potential toxic phase II metabolites. Phytochemicals may influence bioavailability of potential toxic compounds and protect the body against toxic metabolites. benzo[a]pyrene - B[a]P-1-sulfate; B[a]P-3-sulfate B[a]P-3-glucuronide ABC ABC BCRP phytochemicals ABC: ABC-transport proteins Phase 1 und 2: enzymes of phase 1 and 2 Phase 1 Phase 2 ABC enterocyte (Caco-2) phytochemicals benzo[a]pyrene metabolite blood (basal) Page 12

13 Acknowledgment Dr. Bettina Ebert Dr. Lutz Stumkat Yan Li (Ch), Ph.D Roland Büsen,Ph.D Melissa Mock (AUS) Ph.D Fachgruppe 56 des BfR PD Dr. Albrecht Seidel Biochemisches Institut für Umweltkarzinogene, Prof. G. Grimmer-Foundation, Großhansdorf Prof. A. Schinkel, University of Amsterdam, Netherlands Prof. H.R. Glatt DIFE, Rehbrücke, Potsdam Thank you Prof. Dr. Dr. Alfonso Lampen Department of Food Safety Federal Institute for Risk Assessment (BfR), for your attention Page 13