Effects on human health

Polyphenols Several hydroxyl groups on aromatic rings Secondary metabolites (> 10,000) of plants Several classes: phenolic acids, stilbenes, lignans, flavonoids (Largely) present in our food Plant defence Leaves Stems Fruits Interaction with microorganisms Seeds Roots Flowers Color (fruits, flowers)

Effects on human health Cardiovascular diseases Cancers Osteoporosis Polyphenol properties: antioxidant anticarcinogenic vasodilatator antiviral antibacterial anti-inflammatory Inflammatory diseases Neurodegenerative diseases

POLYPHENOLS ET FONCTION INTESTINALE : APPROCHE IN VITRO Y.-J. Schneider,, B. Romier,, T. Sergent, S. Hollebeeck,, J. Van De Walle,, A. During, I. Dupont, A. Joly, L. Ribonnet,, A.-C. Schneider, A. Hendrickx,, P. Beguin & Y. Larondelle Biochimie cellulaire, nutritionnelle & toxicologique Institut des Sciences de la Vie UCL-Louvain-la-Neuve <yjs@uclouvain.be> GP3A

S P Intestinal barrier: physiology Different cell types Crypt cells Neuroendocrine cells Goblet cells Enterocytes M cells Different functions Secretion: fluid, electrolytes Immunity Secretion: hormones, Final digestion and absorption of nutrients Special functions Uptake of particles Excretion Presystemic biotransformations

Intestinal barrier: pathological risks? Intestinal inflammation "Physiological" inflammation Chronic inflammation: Crohn disease, Increased risk of colon cancer Presystemic biotransformations/ transport Cytochrome P450: : phase I Conjugations: phase II Efflux pumps: : phase III Increased risk of colon cancer

Tools to investigate the intestinal function Monolayer Microporous filter Tight junctions Enterocyte Enzymes Upper compartment: apical pole lumen Lower compartment: basolateral pole blood Microvillosities In vivo approach Ex vivo approach Cell culture systems: in vitro model of the human intestinal barrier Caco-2 cell line: human colon adenocarcinoma, differentiates into enterocytes Microporous filter bicameral insert Transporters

Model of ochratoxin A transport OTA MRP2 ph 6.0 OTA diffuses across Caco-2 cells ph gradient binding of OTA to albumin increase absorption Paracellular passage is low but increased by mycotoxins OTA albumin ph 7.4 OTA absorption is limited by MRP2 efflux (but also BCRP) at AP side Berger et al., Toxicol. Lett., 140-141, 465-476, 2003

Effect of polyphenols on OTA transport Caco-2 cells with [ 3 H] OTA: excretion increased by inhibition of MRP2 Polyphenols at realistic food concentrations increase OTA absorption Competition between OTA and polyphenols conjugates? Sergent et al., Toxicol. Lett., 159, 60-70, 2005

NFκB B activation: key of inflammation TNF-α IL-1 LPS MEKK-1 MAPKK MAPK Degradation IKKa p50 P (NFκB) IKKγ IKKβ P IκB p65 p50/p65 Transduction cascades of cytokines or LPS activate NFκB via IκB phosphorylation or MAPKinases Nuclear translocation activation of expression of inflammatory proteins expression Continuous activation chronic intestinal inflammatory diseases TARGET GENES Cytokines (IL-1β, IL-8, ) Enzymes (inos, COX-2, )

Effect of polyphenols on NFκB B activation Δ9 desaturase activity Plasmid 4X-NFkB Treatment Fold increase / 1 IL-1! 7,5 IL -1!gallic acid 7,7 IL -1!ellagic acid IL -1!catechin IL-1!quercetin IL-1!naringenin IL -1!genistein 1,8 7,8 6,5 6,3 15,1 * * Caco-2 cells exposed to IL-1ß activate NFκB (transient transfection/reporter gene) Modulation by polyphenols Comparable results upon activation with TNF-α or LPS IL -1!chrysin IL-1!epicatechin IL -1!resveratrol 1,2 5,1 12 * 0 200 400 600 800 1000 1200 1400 1600 1800 Polyphenols modulate intestinal inflammation R.L.U./ µ g of protein (% of c ontrol) Romier et al., Br.. J. Nutrition, 100, 542-551, 2008

Polyphenols and IκB I phosphorylation P-IκBα Caco-2 cells: ± IL-1ß, -actin ± proteasome 2 inhibitor c 1.8 bc ± [polyphenols] bc 1.6 bc bc b bc 1.4 WB for phosphorylated IκBα b b b 1.2 1 IκBα activation: IL-1ß MG132 0.8 Ellagic acid phosphorylation 0.6 a a 0.4 Chrysin β-actin Relative intensity... 0.2 MG132 IL-1β 0 a - - - GA EA CHRY GEN QUER NAR CAT EPI RESV Romier et al., Br.. J. Nutrition, 100, 542-551, 2008 Polyphenols modulate differently NFκB B activation

Effect of polyphenols on IL-8 secretion IL-8 secretion... (% control) 200 150 100 50 a b a ab a a c Differentiated Caco-2 cells exposed to IL-1ß; ; IL-8 Elisa Increase of secretion by IL-1ß (TNFα or LPS) Modulation by polyphenols 0 - EA CHRY GEN EGCG RESV IL-1β Food compounds modulate intestinal inflammation Romier et al., Br.. J. Nutrition, 100, 542-551, 2008

Effect of polyphenols on MAPKinases Relative intensity Relative intensity Relative intensity 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 3 2.5 2 1.5 1 0.5 0 ERK1/2 - EA CHRY RESV EGCG P38 - EA CHRY RESV EGCG Differentiated Caco-2 cells ± IL-1ß for 24H, ± 50 µm of polyphenols Western blotting for total and phosphorylated MAPKinases Activation of MAPKinases by IL-1ß Different activations of MAPKinases by polyphenols 8 7 SAPK/JNK Relative intensity Relative intensity 6 5 4 3 2 1 0 - EA CHRY RESV EGCG Complex modulation of MAPKinases by polyphenols Romier et al., in press,, 2008

Effect on CYP1A1 induction CYP 1A1: : highly inducible Pollutants interact with AhR transduction cascade Nuclear translocation induction of Cyp1A1 biotransformation activity Possible risk of increased carcinogenicity http://www.stanford stanford.edu/group/whitlock/newresearch.html

CYP1A1 induction: effect of polyphenols pmoles/min./mg prot 40 35 30 25 20 15 10 5 0 / gen: genistein quer: quercetin chrys: chrysin gen 5 gen 50 quer 5 quer 50 chrys 5 chrys 50 Sergent et al., in preparation 3-MC 3-MC gen 5 CYP1A1 activity 3-Methylcholanthrene 3-MC gen 50 3-MC quer 5 3-MC quer 50 3-MC chrys 5 3-MC chrys 50 Benzo-a-pyrene -pyrene BaP BaP gen 5 BaP gen 50 BaP quer 5 BaP quer 50 BaP chrys 5 BaP chrys 50 118.6 ± 21.4 Dioxin TCDD TCDD gen 5 TCDD gen 50 TCDD quer 5 TCDD quer 50 TCDD chrys 5 TCDD chrys 50 Little induction by some polyphenols: : possible agonists of AhR High induction by 3-MC, Bap and TCDD (dioxin) High decrease of Cyp1A1 induction by some polyphenols Preventive effect of PAHs by food constituents?

Bioaccessibility / Bioavailability of polyphenols Bioaccessibilty: : fraction of the ingested polyphenol that is released from the food matrix and available in the gut for its possible absorption Bioavailability: fraction of the ingested polyphenol that is absorbed and available in bloodstream for its utilization by the body.

Bioaccessibility / Bioavailability Polyphenol in food Salivary digestion Gastric digestion Polyphenol Polyphenol Pancreatic digestion Apical medium Cell monolayer on membrane Basolateral medium Polyphenol and its metabolites analyzed by HPLC in cells and media ABSORPTION METABOLISM

Conclusions / Future work Polyphenols are affected,, but also affect the intestinal function Polyphenols modulate transduction cascades and gene expression,, as well as proteins in Caco-2 cells Different effects for different polyphenols Caco-2 cells culture system seems relevant Importance of polyphenols mixtures Intestinal interactions : food f matrix, contaminants, drugs, Mechanistic approaches : transcriptomics,, permeability, Bioaccessibility / bioavailability

Acknowledgments Prof. Hervé Rogez, Univ.. Federal do Para, Brazil Prof. Jean-Louis Habib-Jiwan,, ISV, Be Dr. Vincent Baeten,, CRA-W, Be Dr. Luc Pussemier,, CERVA-CODA, Be Partners of the "Walnut-20" project Fundings from