High throughput metabolic approaches to identify common functionalities of dietary polyphenols

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1 High throughput metabolic approaches to identify common functionalities of dietary polyphenols Augustin SCALBERT Clermont-Ferrand

2 Antioxidant properties Polyphenols Health No dietary recommendations for polyphenols - Most protective polyphenols? - Most protective levels of intake?

3 Polyphenols as antioxidants Reduction of free radicals Scavengers of free radicals (ROS, RNS) HO O R Too low concentrations in tissues - Polyphenols : < 1 µm - Uric acid : µm; Glutathion : 1-10 mm Protective effects not necessarly linked to protection of cell constituents against oxidation

Polyphenols as antioxidants Reduction of free radicals > 300 different

Polyphenol database ABTS + reduction Cell growth inhibition Quercetin

Naringenin Apigenin Chrysin Hesperetin Kaempferol Daidzein Genistin

4 Polyphenols as antioxidants Reduction of free radicals > 300 different polyphenols in food Reducing properties essential for activity? Polyphenol database ABTS + reduction Cell growth inhibition Quercetin Myricetin Genistein Morin Epicatechin Catechin Rutin Luteolin Taxifolin Naringenin Apigenin Chrysin Hesperetin Kaempferol Daidzein Genistin Biochanin A Hesperidin Formononetin (Rice-Evans & Miller, 1996) 0 0,01 0,02 (Kuntz et al., 1999)

5 Polyphenols as antioxidants Reduction of cell signalling proteins HO O Signalling Genes Redox-sensitive protein Metabolism Redox sensitive ligands and signalling pathways Common pathways / genes modulated by all polyphenols? Redox potential of polyphenols + accessibility to the substrate = key parameters?

6 Polyphenols as antioxidants Effects on redox metabolism HO O Metabolism Oxidative stress biomarkers Genes Redox metabolism Redox effects independent of polyphenol reducing properties? Redox metabolism may itself trigger redox sensitive signalling pathways

7 Polyphenols,, more than antioxidants HO O Genes Metabolism Redox metabolism Health Metabolism Various cell signalling pathways and genes modulated by polyphenols NFkB, AP-1, Nrf2, MAPK, PPARγ, AhR, ERα, SREBP Complex metabolic responses

8 Polyphenols Health Compare metabolic responses of different polyphenols Structural requirements for activity? Phenolic moieties essential for activity? High throughput approaches to explore polyphenol effects on metabolism

9 Polyphenol doses in in vitro studies Effects on gene expression (Ludwig et al., 2004) (Choi et al., 2004) (Hernandez-Montes et al., 2006) (Reiterer et al., 2004) (Fuchs et al., 2005) (Kim et al., 2005) (Chao et al., 2005) (Mnjoyan et al., 2003) (Pal et al., 2003) (Gouedard et al., 2004) (Martinez-Florez et al., 2005) (Mullen et al., 2004) (Sarkar et al., 2004) (Richard et al., 2005) (Kim et al., 2005) (Moreno et al., 2003) (Mackenzie et al., 2003) (Yang et al., 1998) (Biswas et al., 2005) (Abate et al., 2005) (Kim et al., 2004) Active polyphenol concentrations (µm) 0, > 5 µm Endothelial cells Aortic smooth muscle cells Hepatocytes Immune cells Lung epithelial cells Fibroblasts β-cells Various polyphenols (catechins, quercetin, luteolin, gallic acid, tyrosol, isoflavones ) Various genes (ICAM-1, VCAM-1, E-selectin, ggcs, GPx, IL-6, cfos, endothelin-1, HMGCoA red., LDL receptor, PON-1, inos, TNFa, COX-2 ) Various cell signalling pathways (NFkB, AP-1, MAPK, PPARg, AhR, Era, SREBP)

10 Polyphenol exposure Human studies Plasma conc.( µmol / L ) Single dose of 50 mg aglycone equivalent Time (hr( hr) Genistein (9) Daidzein (9) Querc Gluc. (7) Rutin (6) Naringin (6) Hesperidin (5) Catechins (12) EGCg (11) Anthocyanins (6) (x) = Number of studies Manach et al. (2004) AJCN Concentrations in plasma most often < 1 µm No evidence of accumulation in tissues

11 Polyphenol doses in in vitro studies Effects on gene expression (Ludwig et al., 2004) (Choi et al., 2004) (Hernandez-Montes et al., 2006) (Reiterer et al., 2004) (Fuchs et al., 2005) (Kim et al., 2005) (Chao et al., 2005) (Mnjoyan et al., 2003) (Pal et al., 2003) (Gouedard et al., 2004) (Martinez-Florez et al., 2005) (Mullen et al., 2004) (Sarkar et al., 2004) (Richard et al., 2005) (Kim et al., 2005) (Moreno et al., 2003) (Mackenzie et al., 2003) (Yang et al., 1998) (Biswas et al., 2005) (Abate et al., 2005) (Kim et al., 2004) Active polyphenol concentrations (µm) 0, Nutritional polyphenol concentrations Endothelial cells Aortic smooth muscle cells Hepatocytes Immune cells Lung epithelial cells Fibroblasts β-cells None of these studies carried out at nutritional concentrations

12 Polyphenol doses in in vitro studies KS483 cells from mice Genistein, 18 days 1 µm Differentiation into osteoblasts µm Differentiation into adipocytes Dang ZC et al., 2003, JBC Different effects at nutritional and pharmacological doses

Polyphenol doses in animal experiments Polyphenol intake (mg/kg diet) 0 1000 2000 3000 4000 5000 Hayek et al., 1997 Quercetin Hayek et al., 1997 Catechin ApoE KO mice Hayek et al., 1997 Fuhrman et al.

13 Polyphenol doses in animal experiments Polyphenol intake (mg/kg diet) Hayek et al., 1997 Quercetin Hayek et al., 1997 Catechin ApoE KO mice Hayek et al., 1997 Fuhrman et al., 2005 Red wine Grape powder Protective Kaplan et al., 2001 Pomegranate juice Waddington et al., 2004 Red wine Jiang et al., 2003 Dihydrodaidzein Atherosclerotic lesions Hishikawa et al., 2005 Miura et al., 2001 Bentzon et al., 2001 Yoshikawa et al., 2001 Zhang et al., 1997 CAPE Tea extract Red wine Detrimental Probucol Probucol Different effects according to the dose Protective at low nutritionnal doses Detrimental at high pharmacological doses

Polyphenols and atherosclerosis Comparison of different polyphenols ApoE KO mice (n = 20) +/- Polyphenols (60-200 mg/kg diet) 2, 6

14 Polyphenols and atherosclerosis Comparison of different polyphenols ApoE KO mice (n = 20) +/- Polyphenols ( mg/kg diet) 2, 6 and 14 weeks Atherosclerotic lesions in aortic root Gene expression in aorta and liver Pangenomic arrays (24,000 oligonucleotides)

15 Catechin and atherosclerosis HO O - + 1,8 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 Athersclerotic lesions p<0,05 About 270 genes down-regulated About 490 genes differentially expressed in the aorta 0 Temoins herosclerotic lesions surface Control Catéchine Catechin 6 wks About 220 genes up-regulated Decrease of the atherosclerotic lesion development Expression of various genes affected in aorta

16 Polyphenols and metabolism beyond gene expression HO O Genes Catechin mrna Proteins Metabolites Metabolome metabolites Rats (n = 6) Diets +/- fat, +/- catechin 1 month Urine LC-ESI-Tof PLS-DA

17 Catechin and high-fat diets A metabolomic study HO O Urine metabolome (PLS-DA) m/z % Fat % catechin 0.2 % catechin 15% Fat 5% Fat No catechin Fat in diet (%) > 200 metabolic markers in urine affected by fat intake Some of these markers corrected by catechin

18 Conclusions Polyphenols, metabolism and health Biological effects of polyphenols should be explored at low nutritional doses Structural determinants of activity still largely unknown Comparison of polyphenols in vivo needed to evaluate their respective effects on health Understanding these differences will be essential to establish dietary recommendations and optimize food composition

19 INRA Clermont-Ferrand Aknowledgments Claudine MANACH Dragan MILENKOVIC Sylvain AUCLAIR André MAZUR Anthony FARDET Rafael LLORACH Poster n 89 Poster n 2 Jean-François MARTIN Estelle PUJOS Centre de Recherche de Clermont-Ferrand / Theix

Flavonoids and their contribution to health: a look at the scientific support

Flavonoids and their contribution to health: a look at the scientific support Frank Hu, MD, PhD Professor of Nutrition and Epidemiology Harvard School of Public Health Professor of Medicine Harvard Medical