The Gut Microbiota Ellagic Acid-Derived Metabolite Urolithin A and Its Sulfate Conjugate Are Substrates for the Drug Efflux Transporter Breast Cancer Resistance Protein (ABCG2/BCRP)

The gut microbiota ellagic acid - derived metabolite urolithin A, and its sulfate conjugate, are substrates for the drug efflux transporter breast cancer resistance protein (ABCG2/BCRP). Journal: Journal of Agricultural and Food Chemistry Manuscript ID: jf-2013-007505 Manuscript Type: Article Date Submitted by the Author: 15-Feb-2013 Complete List of Authors: González-Sarrías, Antonio; CEBAS-CSIC, Food Sci. & Technol. Miguel, Veronica; University of Leon, Department of Biomedical Sciences (Physiology) Merino, Gracia; University of Leon, Department of Biomedical Sciences- Physiology Lucas, Ricardo; Instituto de Investigaciones Químicas, CSIC - Universidad de Sevilla, Química Bioorgánica Morales, Juan; Instituto de Investigaciones Químicas, CSIC - Universidad de Sevilla, Química Bioorgánica Tomás-Barberán, Francisco; CEBAS-CSIC, Alvarez, Ana I.; University of Leon, Department of Biomedical Sciences (Physiology) Espín, Juan C.; CEBAS-CSIC, Food Sci. & Technol. ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 1 The gut microbiota ellagic acid-derived metabolite urolithin A, and its sulfate conjugate, are substrates for the drug efflux transporter breast cancer resistance protein (ABCG2/BCRP). ANTONIO GONZÁLEZ-SARRÍAS†, VERÓNICA MIGUEL‡, GRACIA MERINO‡, RICARDO LUCAS#, JUAN C. MORALES#, FRANCISCO TOMÁS-BARBERÁN†, ANA I. ÁLVAREZ‡, JUAN C. ESPÍN†* †Research Group on Quality, Safety and Bioactivity of Plant Foods, Dept. Food Science and Technology, CEBAS-CSIC, P.O. Box 164, 30100 Campus de Espinardo, Murcia, Spain; ‡Department of Biomedical Sciences-Physiology, Campus Vegazana, University of León, 24071 León, Spain and #Department of Bioorganic Chemistry, Institute of Chemical Research, CSIC- University of Seville, 41092 Seville, Spain. *Corresponding author: Juan Carlos Espín, Research Group on Quality, Safety and Bioactivity of Plant Foods, Dept. Food Science and Technology, CEBAS-CSIC, P.O. Box 164, 30100 Campus de Espinardo, Murcia, Spain. Tel: +34 968 396344. Fax. +34-968 396213. E-mail address: [email protected] RUNNING TITLE: Urolithins and ABCG2/BCRP transporter Page 1 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 2 ABSTRACT 1 The breast cancer resistance protein (BCRP/ABCG2) is a drug efflux transporter that can affect 2 the pharmacological and toxicological properties of many molecules. Urolithins, metabolites 3 produced by the gut microbiota from ellagic acid (EA) and ellagitannins, have been 4 acknowledged with in vivo anti-inflammatory and cancer chemopreventive properties. We here 5 evaluated whether urolithins (Uro-A, -B, -C and -D), their main phase II metabolites Uro-A 6 sulfate, Uro-A glucuronide, and Uro-B glucuronide as well as their precursor EA were substrates 7 for ABCG2/BCRP. Parental and Bcrp1-transduced MDCKII cells were used for active transport 8 assays. Uro-A, and in a lesser extent Uro-A sulfate, showed a significant increase in apically 9 directed translocation in Bcrp1-transduced cells. Bcrp1 did not show affinity for the rest of tested 10 compounds. Data were confirmed for murine, human, bovine and ovine BCRP-transduced 11 subclones as well as with the use of the selective BCRP inhibitor Ko143. The transport inhibition 12 by Uro-A was analyzed by flow cytometry compared to Ko143 using the antineoplastic agent 13 mitoxantrone as a model substrate. Results showed that Uro-A was able to inhibit mitoxantrone 14 transport in a dose-dependent manner. We report here for the first time that Uro-A and its sulfate 15 conjugate are ABCG2/BCRP substrates. Our results suggest that physiologically relevant 16 concentrations of these gut microbiota-derived metabolites could modulate ABCG2/BCRP-17 mediated transport processes and mechanisms of cancer drug resistance. Further in vivo 18 investigations are warranted. 19 20 21 22 KEYWORDS: Urolithin; ellagic acid; ABCG2/BCRP transporters; gut microbiota; Bcrp1-23 MDCKII cells. 24 Page 2 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 3 INTRODUCTION 25 ABCG2/BCRP is an ATP-binding cassette (ABC) transporter that mediates energy-dependent 26 translocation of substrates from cells in the intestine, liver, kidney, mammary gland, etc. across 27 cellular membranes, affecting the pharmacokinetics and disposition of drugs and other 28 compounds such as xenotoxins, endogenous compounds, etc. in tissues, and mediating drug-drug 29 interactions.1,2 The multifunctional behavior of ABCG2/BCRP linked to great substrate 30 specificities for a wide range of compounds supported its important role in homeostatic 31 processes.3,4 In addition, the modulation of ABCG2/BCRP transporters can affect 32 chemotherapeutic treatments by modulating the pharmacokinetic behavior of anticancer drugs.5-7 33 Some in vitro and in vivo studies using BCRP-overexpressing cell lines and/or knockout mice 34 have indicated that plant-derived polyphenols such as flavonoids and the stilbene resveratrol can 35 interact directly with BCRP by modulating both its transport function and ATPase activity.8-11 36 Recently, the important role of ABCG2/BCRP in the transport of glucuronide and sulfate 37 conjugates of polyphenols including the flavonoids naringenin, genistein and daidzein,12-14 as 38 well as resveratrol conjugates15 has been described. 39 Urolithins are dibenzopyran-6-one derivatives produced from ellagitannins and ellagic acid 40 (EA) by the gut microbiota through the loss of one lactone ring present in EA and successive 41 removals of hydroxyl groups16-18 (Figure 1). Urolithins have been found in plasma, urine, feces 42 and tissues in a number of animals including the pig,19 rat,20-22 mouse,23 beef cattle,24 etc. 43 Urolithins are also produced by humans, and their occurrence has been reported in plasma, feces, 44 urine,16,18,25,26 and also prostate tissue27 after the intake of ellagitannin-containing foods such as 45 pomegranates, strawberries, raspberries, walnuts, and oak-aged wines. Phase II derived 46 Page 3 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 4 conjugates of urolithins reach the plasma and systemic organs at low micromolar concentrations, 47 whereas high concentrations of the aglycone forms can be found in the gut.19,22 48 In the last decade, after our first report about the occurrence of urolithins in humans,16 49 numerous in vitro and in vivo studies have shown a wide range of biological activities of 50 urolithins, mainly attributed to Uro-A, such as anticancer,22,23,28-30 anti-inflammatory ,31-35 anti-51 bacterial,36 and estrogenic/anti-estrogenic.37 However, the possible role of ABC transporters in 52 the bioavailability of urolithins remains unknown. 53 Our aim was to evaluate whether ellagic acid (EA), urolithins A, B C and D (Uro-A, Uro-B, 54 Uro-C and Uro-D) and their main glucuronide and sulfate derivates (Uro-A glucuronide, Uro-B 55 glucuronide, and Uro-A sulfate) might be ABCG2/BCRP substrates, and/or inhibitors. 56 57 MATERIALS AND METHODS 58 Materials. Ellagic acid (EA) was purchased from Sigma-Aldrich (St. Louis, USA). 59 Urolithin A (3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one; Uro-A) and urolithin B (3-hydroxy-60 6H-dibenzo[b,d]pyran-6-one; Uro-B) were synthesized as described previously.38 Uro B 61 glucuronide (Uro-B glur) was prepared according to Lucas et al. (2009).39 Urolithin A 62 glucuronide (Uro-A glur) and Urolithin A sulfate (Uro-A sulphate) were prepared as a mixture of 63 regioisomers (Figure 2). Experimental procedure and 1H-NMR data for the synthesis of Uro-A 64 and Uro-A sulfate are detailed in Supplementary Methods. Briefly, Uro-A was first mono-65 protected with a silyl protecting group (Figure 2A). Random TBDMS-protection (tert-66 butyldimethylsilyl)40 of Uro-A and subsequent chromatographic separation afforded the two 67 possible mono-phenolic derivatives 1 and 2 as a regioisomeric mixture (1:1) and the double 68 silylated Uro-A. The glycosylation reaction of acceptors 1 and 2 with glucuronosyl donor 3 was 69 Page 4 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 5 performed using the same conditions described previously39 to give compounds 4 and 5 as a 70 regioisomeric mixture (83%). Ester hydrolysis and deprotection of acetyl and TBDMS groups 71 were carried out in one step by using K2CO3 and KF in a methanol-water solution. Reverse phase 72 purification of the crude afforded a 1:1 regioisomeric mixture of Uro-A 3-glucuronide and Uro-A 73 8-glucuronide (Figure 2A). Uro-A sulfate was also prepared as a mixture of regioisomers 74 following a similar strategy to the one used for the glucuronate derivatives (Figure 2B). Sulfation 75 of the mono-silylated urolithin derivatives 1 and 2 was carried out with SO3●NMe3 as sulfating 76 reagent, NEt3 as base, and acetonitrile as solvent at 100 ºC under microwave radiation for 20 min. 77 The reaction afforded the sulfated 1:1 mixture of urolithin derivatives 6 and 7 in 92 % yield 78 (Figure 2B). Final silyl deprotection with KF and reverse phase purification gave a 1:1 mixture 79 of Uro-A 3-sulfate and Uro-A 8-sulfate (73% yield). Urolithin C (3,7,8-trihydroxy-6H-80 dibenzo[b,d]pyran-6-one, Uro-C) and urolithin D (2,3,7,8-tetrahydroxy-6H-dibenzo[b,d]pyran-6-81 one, Uro-D) were purchased from Dalton Pharma Services (Toronto, Canada). Purity was higher 82 than 95% in all tested compounds. Dimethyl sulfoxide (DMSO), diethyl-ether and HPLC 83 reagents, formic acid and acetonitrile (ACN), were obtained from Panreac (Barcelona, Spain). 84 Methanol (MeOH) was from Lab-Scan (Gliwice, Poland). Ko143 was purchased from Tocris 85 (Bristol, United Kingdom). Ultrapure Millipore water was used for all the experiments. 86 87 Cell Lines and Cell Culture Conditions. MDCKII cells (Mardin-Darby canine kidney) 88 and their human BCRP-transduced and murine Bcrp1-transduced sub-clones were kindly 89 provided by Dr. A.H. Schinkel, Netherlands Cancer Institute (Amsterdam). MDCKII cells stably 90 transduced with bovine and ovine variants of ABCG2 have recently been generated by the 91 research group.41,42 Culture conditions were as previously described.43 The cells were cultured in 92 Page 5 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 6 Dulbecco’s modified Eagle’s medium (DMEM) containing GlutaMAX (Life Technologies, Inc., 93 Madrid, Spain) and supplemented with penicillin (50 U/mL), streptomycin (50 μg/mL) and 10% 94 (v/v) fetal calf serum (MP Biomedicals; Cambridge, UK) at 37 °C in the presence of 5% CO2. 95 The cells were trypsinized every 3–4 days for sub-culturing. All of the test samples were 96 solubilized in DMSO (<0.5 % in the culture medium) and were filter sterilised (0.2 μm) prior to 97 addition to the culture media. Control cells were also run in parallel and subjected to the same 98 changes in medium with a 0.5 % DMSO. 99 100 Transport studies. Transepithelial transport assays using Transwell plates were carried out 101 as described previously9,41 with minor modifications. Cells were seeded on microporous 102 polycarbonate membrane filters (3.0 μm pore size, 24 mm diameter; Transwell 3414; Costar) at a 103 density of 1.0 × 106 cells per well. Cells were grown for 3 days, and the medium was replaced 104 every day. Transepithelial resistance was measured in each well using a Millicell ERS ohmmeter 105 (Millipore). Wells registering a resistance of 200 Ω or greater, after correcting for the resistance 106 obtained in blank control wells, were used in the transport experiments. The measurement was 107 repeated at the end of the experiment to check the tightness of the monolayer. 108 Before the start of the experiment, medium on both sides of the monolayer was replaced with 109 2 mL of Optimem medium (Life Technologies), without serum. After 2 h pre-incubation, the 110 experiment was started by replacing the medium in either the apical or basolateral compartment 111 with fresh Optimem medium containing 20 M of the compound tested (except the preliminary 112 screening which was performed at 10 M). Aliquots of 100 μL were taken from the opposite 113 compartment at 2 and 4 h, and stored at −20 °C until LC-MS analysis. In the co-treatments 114 Page 6 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 7 assays, the inhibitor Ko143 (1 μM) was added 2 h before each compound and remained during all 115 experiment. Experiments were carried out in triplicate. 116 117 Accumulation assays. In vitro accumulation assays were carried out as described 118 previouslyusing mitoxantrone (MXR, 10 M) as fluorescent substrate.44 In brief, subconfluent 119 cultures were used after 36 h from seeding. Cells were incubated in Optimem medium with or 120 without Ko143 inhibitor (1 M) or EA, and urolithins A, B, C and D at different concentrations 121 for 60 min before the addition of MXR. Accumulation of MXR was allowed for 1 h at 37°C. 122 Then, cells were washed, trypsinized, collected and resuspended in PBS with 2.5% fetal calf 123 serum. Relative cellular accumulation of MXR of at least 5,000 cells was determined by flow 124 cytometry using a FACSCalibur cytometer (BD Biosciences, NJ, USA). Samples were gated on 125 forward scatter vs. side scatter to exclude cell debris and clumps. Excitation and emission 126 wavelengths for MXR were 635 and 650 nm, respectively. The fluorescence of the accumulated 127 substrate in tested populations was quantified from histogram plots using the median of 128 fluorescence (MF). At least three independent experiments were performed. 129 BCRP inhibition increases accumulation of MXR in Bcrp1/BCRP-transduced cells and thus 130 increases MF. Possible background fluorescence of urolithins was checked in appropriate 131 channels, but the fluorescence was negligible. Flow cytometry data were processed and analyzed 132 using WinMDI version 2.8 software. 133 Percentage of inhibition was calculated according to the following equation: % inhibition = 134 (MF with tested compound in BCRP cells-MF without tested compound in BCRP cells)/(MF 135 with inhibitor Ko 143 in BCRP cells -MF without tested compound in BCRP cells) x 100. 136 Page 7 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 8 Sample processing for the analysis of urolithins and their derivates. Cell media 137 after transport studies was processed as described elsewhere.34 Briefly, ACN (100 L) was added 138 to 100 L of culture media, vortexed and centrifuged at 16,435×g for 10 min. The supernatant 139 was then concentrated in a Speedvac® concentrator Savant SPD121P (Thermo Scientific, 140 Alcobendas, Spain) and the residue re-dissolved in 100 L of MeOH and filtered (0.45 m) 141 before analysis by LC-MS using the conditions described below. 142 143 LC-MS analysis. Aliquots of processed cell media were analyzed using a LC-MS system 144 (1200 Series, Agilent Technologies, Madrid, Spain). The HPLC system was equipped with a 100 145 × 3 mm i.d., 2.7 m, reverse phase C18 column (Poroshell 120, Agilent) and a single quadrupole 146 mass detector in series (6120 Quadrupole, Agilent). Water:formic acid (99:1, v/v) and ACN were 147 used as mobile phases A and B, respectively, with a flow rate of 0.5 mL/min. The linear gradient 148 started with 5% of solvent B in solvent A, reaching 18% solvent B at 7 min, 28% at 17 min, 50% 149 from 22 min, and 90% at 27 min which was maintained up to 29 min. The initial conditions were 150 re-established at 29 min and kept under isocratic conditions up to 33 min. Injections (5 L) were 151 made for each sample at 25 ºC. The mass detector was a quadrupole mass spectrometer equipped 152 with an ESI system (capillary voltage, 3.5Kv; dry temperature, 350 ºC; drying gas flow, 9 L/min; 153 nebulizer pressure, 40 psi). MS spectra were measured in selective ion monitoring (SIM) mode 154 using [M-H] ions for measuring all tested compounds. MS data were acquired in the negative 155 ionization mode. Identification of all tested compounds was carried out by direct comparison 156 (UV spectra and MS) with available standards and confirmed by their spectral properties, 157 molecular mass and fragmentation pattern. Calibration curves were obtained for each tested 158 Page 8 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 15 ABBREVIATIONS USED 290 ABC, ATP-binding cassette; CAN, acetonitrile; BCRP, breast cancer resistance protein; cDNA, 291 complementary deoxyribonucleic acid; DIPEA, N,N-diisopropylethylamine; DMSO, dimethyl 292 sulfoxide; EA, ellagic acid; Glur, glucuronide; HPLC, High-performance liquid chromatography; 293 MDCKII, Mardin-Darby canine kidney; MDR, multidrug resistance; MeOH, methanol; MRP, 294 multidrug resistant protein; MXR, mitoxantrone; OH-, hydroxyl group; SD, standard deviation; 295 SIM, selective ion monitoring; SAR; structure-activity relationship; TBDMS, tert-296 butyldimethylsilyl; TNF- Tumor necrosis factors-alpha; Uro-, urolithin. 297 298 FUNDING INFORMATION 299 This work was funded by the Projects CICYT ALG2011-22447, AGL2012-31116, 300 Consolider Ingenio 2010, CSD2007-00063 (Fun-C-Food), and Fundación Seneca de la Región de 301 Murcia, Spain (Grupo de Excelencia GERM 06 04486 and 05556/PI/04). 302 303 CONFLICT OF INTEREST 304 The authors declare no competing financial interest. 305 306 SUPPORTING INFORMATION AVAILABLE 307 This material is available free of charge via the Internet at http://pubs.acs.org. 308 Supplementary Methods. Experimental procedure for the synthesis of Uro-A 309 glucuronide and Uro-A sulfate. 310 311 312 Page 15 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 16 REFERENCES 313 (1) Borst, P.; Elferink, R. O. Mammalian ABC transporters in health and disease. Annu. Rev. 314 Biochem. 2002, 71, 537–592. 315 (2) Glavinas, H.; Krajcsi, P.; Cserepes, J.; Sarkadi, B. The role of ABC transporters in drug 316 resistance, metabolism and toxicity. Curr. Drug Deliv. 2004, 1, 27–42. 317 (3) Krishnamurthy, P.; Schuetz, J. D. Role of ABCG2/BCRP in biology and medicine. Annu. 318 Rev. Pharmacol. Toxicol. 2006, 46, 381–410. 319 (4) Glavinas, H.; Kis, E.; Pál, A.; Kovács, R.; Jani, M.; Vági, E.; Molnár, E.; Bánsághi, S.; 320 Kele, Z.; Janáky, T.; Báthori, G.; von Richter, O.; Koomen, G. J.; Krajcsi, P. ABCG2 (breast 321 cancer resistance protein/mitoxantrone resistance-associated protein) ATPase assay: a useful tool 322 to detect drug-transporter interactions. Drug Metab. Dispos. 2007, 35, 1533–1542. 323 (5) Huang, Y.; Sadée, W. Membrane transporters and channels in chemoresistance and -324 sensitivity of tumor cells. Cancer Letters 2006, 239, 168–182. 325 (6) de Wolf, C.; Jansen, R.; Yamaguchi, H.; de Haas, M.; van de Wetering, K.; Wijnholds, J.; 326 Beijnen, J.; Borst, P. Contribution of the drug transporter ABCG2 (breast cancer resistance 327 protein) to resistance against anticancer nucleosides. Mol. Cancer Ther. 2008, 7, 3092–3102. 328 (7) Fukuda, Y.; Schuetz, J. D. ABC transporters and their role in nucleoside and nucleotide 329 drug resistance. Biochem. Pharmacol. 2012, 83, 1073-1083. 330 (8) van de Wetering, K.; Burkon, A.; Feddema, W.; Bot, A.; de Jonge, H.; Somoza, V.; Borst, 331 P. Intestinal breast cancer resistance protein (BCRP)/Bcrp1 and multidrug resistance protein 3 332 (MRP3)/Mrp3 are involved in the pharmacokinetics of resveratrol. Mol. Pharmacol. 2009, 75, 333 876-885. 334 Page 16 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 17 (9) Merino, G.; Pérez, M.; Real, R.; Egido, E.; Prieto, J. G.; Álvarez, A. I. In vivo inhibition 335 of BCRP/ABCG2 mediated transport of nitrofurantoin by the isoflavones genistein and daidzein: 336 A comparative study in Bcrp1(−/−) mice. Pharm. Res. 2010, 27, 2098–2105 337 (10) Álvarez, A. I.; Real, R.; Pérez, M.; Mendoza, G.; Prieto, J. G.; Merino, G. Modulation of 338 the activity of ABC transporters (P-glycoprotein, MRP2, BCRP) by flavonoids and drug 339 response. J. Pharm. Sci. 2010, 99, 598-617. 340 (11) Li, Y.; Lu, J.; Paxton, J. W. The role of ABC and SLC transporters in the 341 pharmacokinetics of dietary and herbal phytochemicals and their interactions with xenobiotics. 342 Curr. Drug Metab. 2012, 13, 624-639. 343 (12) Álvarez, A.I.; Vallejo, F.; Barrera, B.; Merino, G.; Prieto, J. G.; Tomás-Barberán, F. A.; 344 Espín, J. C. Bioavailability of the glucuronide and sulfate conjugates of genistein and daidzein in 345 breast cancer resistance protein 1 knockout mice. Drug Metab. Dispos. 2011, 39, 2008-2012. 346 (13) Pérez, M.; Otero, J. A.; Barrera, B.; Prieto, J.G.; Merino, G.; Álvarez, A. I. Inhibition of 347 ABCG2/BCRP transporter by soy isoflavones genistein and daidzein: Effect on plasma and milk 348 levels of danofloxacin in sheep. Vet. J. 2012, pii: S1090-0233(12)00397-8. 349 (14) Weing, J.; Hu, M. Mutual interactions between flavonoids and enzymatic and transporter 350 elements responsible for flavonoid disposition via phase II metabolic pathways. RSC Advances 351 2012, 2, 7948-7963. 352 (15) Alfaras. I.; Pérez, M.; Juan, M. E.; Merino, G.; Prieto, J. G.; Planas, J. M.; Álvarez, A. I. 353 Involvement of breast cancer resistance protein (BCRP1/ABCG2) in the bioavailability and 354 tissue distribution of trans-resveratrol in knockout mice. J. Agric. Food Chem. 2010, 58, 4523-355 4528. 356 (16) Cerdá, B.; Espín, J. C.; Parra, S.; Martínez, P.; Tomás-Barberán, F. A. The potent in vitro 357 antioxidant ellagitannins from pomegranate juice are metabolised into bioavailable but poor 358 Page 17 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 18 antioxidant hydroxy-6H-dibenzopyran-6-one derivatives by the colonic microflora of healthy 359 humans. Eur. J. Nutr. 2004, 43, 205-220. 360 (17) Cerdá, B.; Periago, P. M.; Espín, J. C.; Tomás-Barberán, F. A. Identification of urolithin 361 A as a metabolite produced by human colon microflora from ellagic acid and related compounds 362 J. Agric. Food Chem. 2005, 53, 5571– 5576 363 (18) Cerdá, B.; Tomás-Barberán, F. A.; Espín, J. C. Metabolism of antioxidant and 364 chemopreventive ellagitannins from strawberries, raspberries, walnuts, and oak-aged wine in 365 humans: identification of biomarkers and individual variability. J. Agric. Food Chem. 2005; 53, 366 227-235. 367 (19) Espín, J. C.; González-Barrio, R.; Cerdá, B.; López-Bote, C.; Rey, A. I.; Tomás-Barberán, 368 F. A. Iberian pig as a model to clarify obscure points in the bioavailability and metabolism of 369 ellagitannins in humans J. Agric. Food Chem. 2007, 55, 10476– 10485. 370 (20) Cerdá, B.; Llorach, R.; Cerón, J. J.; Espín, J. C.; Tomás-Barberán, F. A. Evaluation of the 371 bioavailability and metabolism in the rat of punicalagin, an antioxidant polyphenol from 372 pomegranate juice. Eur. J. Nutr. 2003, 42, 18-28. 373 (21) Ito, H.; Iguchi, A.; Hatano, T. Identification of urinary and intestinal bacterial metabolites 374 of ellagitannin geraniin in rats. J. Agric. Food Chem. 2008, 56, 393–400. 375 (22) González-Sarrías, A.; Azorín-Ortuño, M.; Yáñez-Gascón, M. J.; Tomás-Barberán, F. A.; 376 García-Conesa, M. T.; Espín, J. C. Dissimilar in vitro and in vivo effects of ellagic acid and its 377 microbiota-derived metabolites, urolithins, on the cytochrome P450 1A1. J. Agric. Food Chem. 378 2009, 57, 5623-5632. 379 (23) Seeram, N. P.; Aronson, W. J.; Zhang, Y.; Henning, S. M.; Moro, A.; Lee, R. P.; 380 Sartippour, M.; Harris, D. M.; Rettig, M.; Suchard, M. A.; Pantuck, A. J.; Belldegrun, A.; Heber, 381 Page 18 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 19 D. Pomegranate ellagitannin-derived metabolites inhibit prostate cancer growth and localize to 382 the mouse prostate gland. J. Agric. Food Chem. 2007, 55, 7732-7737. 383 (24) González-Barrio, R.; Truchado, P.; Ito, H.; Espín, J. C.; Tomás-Barberán, F. A. UV and 384 MS identification of urolithins and nasutins, the bioavailable metabolites of ellagitannins and 385 ellagic acid in different mammals J. Agric. Food Chem. 2011, 59, 1152– 1162. 386 (25) Cerdá, B.; Soto, C.; Albaladejo, M. D.; Martínez, P.; Sánchez-Gascón, F.; Tomás-387 Barberán, F.; Espín, J. C. Pomegranate juice supplementation in chronic obstructive pulmonary 388 disease: a 5-week randomized, double-blind, placebo-controlled trial. Eur. J. Clin. Nutr. 2006, 389 60, 245-253. 390 (26) Truchado, P.; Larrosa, M.; García-Conesa, M. T.; Cerdá, B.; Vidal-Guevara, M. L.; 391 Tomás-Barberán, F. A.; Espín, J. C. Strawberry processing does not affect the production and 392 urinary excretion of urolithins, ellagic acid metabolites, in humans. J. Agric. Food Chem. 2011, 393 60, 5749-5754. 394 (27) González-Sarrías, A.; Giménez-Bastida, J. A.; García-Conesa, M. T.; Gómez-Sánchez, M. 395 B.; García-Talavera, N. V.; Gil-Izquierdo, A.; Sánchez-Alvarez, C.; Fontana-Compiano, L. O.; 396 Morga-Egea, J. P.; Pastor-Quirante, F. A.; Martínez-Díaz, F.; Tomás-Barberán, F. A.; Espín, J.C. 397 Occurrence of urolithins, gut microbiota ellagic acid metabolites and proliferation markers 398 expression response in the human prostate gland upon consumption of walnuts and pomegranate 399 juice. Mol. Nutr. Food Res. 2010, 54, 311– 322 400 (28) González-Sarrías, A.; Espín, J. C.; Tomás-Barberán, F. A.; García-Conesa, M. T. Gene 401 expression, cell cycle arrest and MAPK signalling regulation in Caco-2 cells exposed to ellagic 402 acid and its metabolites, urolithins. Mol. Nutr. Food Res. 2009, 53, 686-698. 403 Page 19 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 20 (29) Sharma, M.; Li, L.; Celver, J.; Killian, C.; Kovoor, A.; Seeram, N. P. Effects of fruit 404 ellagitannin extracts, ellagic acid, and their colonic metabolite, urolithin A, on Wnt signaling. J. 405 Agric. Food Chem. 2010, 58, 3965-3969. 406 (30) Kasimsetty, S. G.; Bialonska, D.; Reddy, M. K.; Ma, G.; Khan, S. I.; Ferreira, D. Colon 407 cancer chemopreventive activities of pomegranate ellagitannins and urolithins. J. Agric. Food 408 Chem. 2010, 58, 2180-2187. 409 (31) Larrosa, M.; González-Sarrías, A.; Yáñez-Gascón, M.J.; Selma, M. V.; Azorín-Ortuño, 410 M.; Toti, S.; Tomás-Barberán, F.; Dolara, P.; Espín, J. C. Anti-inflammatory properties of a 411 pomegranate extract and its metabolite urolithin-A in a colitis rat model and the effect of colon 412 inflammation on phenolic metabolism. J. Nutr. Biochem. 2010; 21, 717-725. 413 (32) González-Sarrías, A.; Larrosa, M.; Tomás-Barberán, F. A.; Dolara, P.; Espín, J. C. NF-414 kappaB-dependent anti-inflammatory activity of urolithins, gut microbiota ellagic acid-derived 415 metabolites, in human colonic fibroblasts. Br. J. Nutr. 2010; 104, 503-512. 416 (33) Verzelloni, E.; Pellacani, C.; Tagliazucchi, D.; Tagliaferri, S.; Calani, L.; Costa, L. G.; 417 Brighenti, F.; Borges, G.; Crozier, A.; Conte, A.; Del Rio, D. Antiglycative and neuroprotective 418 activity of colon-derived polyphenol catabolites. Mol. Nutr. Food Res. 2011, 55, S35-S43. 419 (34) Giménez-Bastida, J. A.; Larrosa, M.; González-Sarrías, A.; Tomás-Barberán, F.; Espín, J. 420 C.; García-Conesa, M. T. Intestinal Ellagitannin Metabolites Ameliorate Cytokine-Induced 421 Inflammation and Associated Molecular Markers in Human Colon Fibroblasts. J. Agric. Food 422 Chem. 2012, 60, 8866-8876. 423 (35) Giménez-Bastida, J. A.; González-Sarrías, A.; Larrosa, M.; Tomás-Barberán, F.; Espín, J. 424 C.; García-Conesa, M. T. Ellagitannin metabolites, urolithin A glucuronide and its aglycone 425 urolithin A, ameliorate TNF-α-induced inflammation and associated molecular markers in human 426 aortic endothelial cells. Mol. Nutr. Food Res. 2012, 56, 784-796. 427 Page 20 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 21 (36) Giménez-Bastida, J. A.; Truchado, P.; Larrosa, M.; Espín, J. C.; Tomás-Barberán, F. A.; 428 Allende, A.; García-Conesa, M. T. Urolithins, ellagitannin metabolites produced by colon 429 microbiota, inhibit Quorum Sensing in Yersinia enterocolitica: Phenotypic response and 430 associated molecular changes. Food Chem. 2012, 132, 1465-1474. 431 (37) Larrosa, M.; González-Sarrías, A.; García-Conesa, M. T.; Tomás-Barberán, F. A.; Espín, 432 J. C. Urolithins, ellagic acid-derived metabolites produced by human colonic microflora, exhibit 433 estrogenic and antiestrogenic activities. J. Agric. Food Chem. 2006, 54, 1611-1620. 434 (38) Bialonska, D.; Kasimsetty, S. G.; Khan, S. I.; Ferreira, D. Urolithins, intestinal microbial 435 metabolites of Pomegranate ellagitannins, exhibit potent antioxidant activity in a cell-based 436 assay. J. Agric. Food Chem. 2009, 57, 10181-10186. 437 (39) Lucas, R.; Alcantara, D.; Morales, J. C. A concise synthesis of glucuronide metabolites of 438 urolithin-B, resveratrol, and hydroxytyrosol. Carbohydr. Res. 2009, 344, 1340-1346. 439 (40) Tanis, V. M.; Moya, C.; Jacob, R. S.; Little, R. D. Synthesis and evaluation of the 440 bioactivity of simplified analogs of the seco-pseudopterosins; progress toward determining a 441 pharmacophore. Tetrahedron 2008, 64, 10649-10663. 442 (41) Real, R.; González-Lobato, L.; Baro, M. F.; Valbuena, S.; de la Fuente, A.; Prieto, J. G.; 443 Álvarez, A. I.; Marqués, M. M.; Merino, G. Analysis of the effect of the bovine ABCG2 SNP 444 Y581S on transcellular transport of veterinary drugs using new cell culture models. J. Anim. Sci. 445 2011, 89, 4325–4338. 446 (42) González-Lobato, L.; Real, R.; Herrero, D.; de la Fuente, A.; Prieto, J. G.; Márques, M. 447 M.; Álvarez, A. I.; Merino, G. Identification of substrates and inhibitors of the ovine and bovine 448 adenosine triphosphate-binding cassette transporter G2: interaction with veterinary drugs, 449 micronutrients and natural products. Submitted to Journal of Animal Science. 450 Page 21 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 22 (43) Jonker, J. W.; Smit, J. W.; Brinkhuis, R. F.; Maliepaard, M.; Beijnen, J. H.; Schellens, J. 451 H.; Schinkel, A. H. Role of breast cancer resistance protein in the bioavailability and fetal 452 penetration of topotecan. J. Natl. Cancer Inst. 2000, 92, 1651–1656. 453 (44) Pavek, P.; Merino, G.; Wagenaar, E.; Bolscher, E.; Novotna, M.; Jonker, J. W.; Schinkel, 454 A. H. Human breast cancer resistance protein: interactions with steroid drugs, hormones, the 455 dietary carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, and transport of 456 cimetidine. J. Pharmacol. Exp. Ther. 2005, 312, 144-152. 457 (45) Zhang, S.; Yang, X.; Morris, M. E. Combined effects of multiple flavonoids on breast 458 cancer resistance protein (ABCG2)-mediated transport. Pharm. Res. 2004, 21, 1263–1273. 459 (46) Larrosa, M.; García-Conesa, M.T.; Espín, J.C.; Tomás-Barberán, F.A. Ellagitannins, 460 ellagic acid and vascular health. Mol. Asp. Med. 2010, 31, 513-39. 461 (47) Ishimoto, H.; Shibata, M.; Myojin, Y.; Ito, H.; Sugimoto, Y.; Tai, A.; Hatano, T. In vivo 462 anti-inflammatory and antioxidant properties of ellagitannin metabolite urolithin A. Bioorg. Med. 463 Chem. Lett. 2011, 21, 5901-5904. 464 (48) Ahmed-Belkacem, A.; Pozza, A.; Munoz-Martinez, F.; Bates, S. E.; Castanys, S.; 465 Gamarro, F.; Di Pietro, A.; Perez-Victoria, J. M. Flavonoid structure-activity studies identify 6-466 prenylchrysin and tectochrysin as potent and specific inhibitors of breast cancer resistance protein 467 ABCG2. Cancer Res. 2005, 65, 4852–4860. 468 (49) Pick, A.; Müller, H.; Mayer, R.; Haenisch, B.; Pajeva, I. K.; Weigt, M.; Bönisch, H.; 469 Müller, C. E.; Wiese, M. Structure-activity relationships of flavonoids as inhibitors of breast 470 cancer resistance protein (BCRP). Bioorg. Med. Chem. 2011, 19, 2090-2102. 471 (50) van de Wetering, K.; Sapthu, S. ABCG2 functions as a general phytoestrogen sulfate 472 transporter in vivo. FASEB J. 2012, 26, 4014-4024. 473 Page 22 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 23 (51) Tamaki, H.; Satoh, H.; Hori, S.; Ohtani, H.; Sawada, Y. Inhibitory effects of herbal 474 extracts on breast cancer resistance protein (BCRP) and structure-inhibitory potency relationship 475 of isoflavonoids. Drug Metab. Pharmacokinet. 2010, 25, 170-179. 476 (52) González-Lobato, L.; Real, R.; Prieto, J. G.; Álvarez, A. I.; Merino, G. Differential 477 inhibition of murine Bcrp1/Abcg2 and human BCRP/ABCG2 by the mycotoxin fumitremorgin 478 C. Eur. J. Pharmacol. 2010, 644, 41-48. 479 (53) Otero, J. A.; Real, R.; de la Fuente, A.; Prieto, J. G.; Marques, M.; Álvarez, A. I.; Merino, 480 G. The bovine ATP-binding cassette transporter ABCG2 Y581S single nucleotide polymorphism 481 increases milk secretion of the fluoroquinolone danofloxacin. Drug Metab. Dispos. 2012, 482 doi:10.1124/dmd.112.049056. 483 (54) Ang, G.; Morris, M. E. Effects of the isoflavonoid biochanin A on the transport of 484 mitoxantrone in vitro and in vivo. Biopharm. Drug Dispos. 2010, 31, 340-350. 485 (55) González-Barrio, R.; Truchado, P.; García-Villalba, R.; Hervás, G.; Frutos, P.; Espín, J. 486 C.; Tomás-Barberán, F. A. Metabolism of oak leaf ellagitannins and urolithin production in beef 487 cattle. J. Agric. Food Chem. 2012, 60, 3068-3077. 488 (56) van de Wetering, K.; Feddema, W.; Helms, J. B.; Brouwers, J. F.; Borst, P. Targeted 489 metabolomics identifies glucuronides of dietary phytoestrogens as a major class of MRP3 490 substrates in vivo. Gastroenterology 2009, 137, 1725-1735. 491 492 493 494 Page 23 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 24 FIGURE CAPTIONS 495 Figure 1. Chemical structures of EA, Uro-D, Uro-C, Uro-A, Uro-B. The arrows designate the 496 sequence of metabolic steps carried out by the gut microbiota. 497 498 Figure 2. (A) Synthesis of Uro-A glucuronates; a) TBDMSTfO, DIPEA (N,N-499 diisopropylethylamine), CH2Cl2; b) BF3●OEt2; CH2Cl2, glucuronate donor 3; c) K2CO3, KF, 500 MeOH-H2O (5:1). (B) Synthesis of Uro-A sulfates; d) SO3●NMe3, NEt3, CH3CN, 100 ºC, 20 min, 501 MW; e) KF, MeOH. 502 503 Figure 3. Transepithelial transport of tested compounds (10 M) in (A) parental MDCKII cells 504 and in (B) their murine Bcrp1-transduced derivatives. The experiment was started with the 505 addition of each compound to one compartment (basolateral or apical). After 2 and 4 h, the 506 percentage of each compound appearing in the opposite compartment was measured by LC-MS 507 and plotted. Data are expressed as mean values ± SD (n = 3). (○) Translocation from the 508 basolateral to the apical compartment; (●) translocation from the apical to the basolateral 509 compartment. 510 511 Figure 4. Transepithelial transport of Uro-A (20 M) in parental MDCKII cells, and in their 512 murine Bcrp1-, and human, bovine and ovine BCRP-transduced subclones in the absence or 513 presence of Ko143 (1 M). The experiment was carried out as indicated in Figure 2. Data are 514 expressed as mean values ± SD (n = 3). ○, Translocation from the basolateral to the apical 515 compartment; ●, translocation from the apical to the basolateral compartment. 516 517 Page 24 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 31 Figure 6. Uro-A (M) % inhibition compared to 100% Ko143 inhibition 0 10 20 30 40 50 60 70 10 20 50 100 Bcrp1 BCRP Page 31 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry (Breast cancer resistance protein transporter) Basal side (blood) R1= OH and R2= OH Urolithin A R1or R2= OH, and R1or R2= Sulfate Urolithin A sulfate Apical side (lumen) OO R2 R1 ABCG2/BCRP (Drug efflux) Graphical Abstract The gut microbiota ellagic acid-derived metabolite urolithin A, and its sulfate conjugate, are substrates for the drug efflux transporter breast cancer resistance protein (ABCG2/BCRP). Page 32 of 32 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry