Urolithins, Ellagic Acid-Derived Metabolites Produced by Human Colonic Microflora, Exhibit Estrogenic and Antiestrogenic Activities MAR LARROSA,ANTONIO GONZA Ä LEZ-SARRI ÄAS,MARI ÄA TERESA GARCI ÄA-CONESA, FRANCISCO A. TOMA Ä S-BARBERA Ä N, AND JUAN CARLOS ESPI ÄN* Research Group on Quality, Safety and Bioactivity of Plant Foods. Department of Food Science and Technology, CEBAS-CSIC, P.O. Box 164, 30100 Campus de Espinardo, Murcia, Spain Urolithins A and B (hydroxy-6H-dibenzo[b,d]pyran-6-one derivatives) are colonic microflora metabolites recently proposed as biomarkers of human exposure to dietary ellagic acid derivatives. Molecular models suggest that urolithins could display estrogenic and/or antiestrogenic activity. To this purpose, both urolithins and other known phytoestrogens (genistein, daidzein, resveratrol, and enterolactone) were assayed to evaluate the capacity to induce cell proliferation on the estrogen-sensitive human breast cancer MCF-7 cells as well as the ability to bind to R- and -estrogen receptors. Both urolithins A and B showed estrogenic activity in a dose-dependent manner even at high concentrations (40 µM), without antiproliferative or toxic effects, whereas the other phytoestrogens inhibited cell proliferation at high concentrations. Overall, urolithins showed weaker estrogenic activity than the other phytoestrogens. However, both urolithins displayed slightly higher antiestrogenic activity (antagonized the growth promotion effect of 17--estradiol in a dose-dependent manner) than the other phytoestrogens. The IC 50 values for the ERR and ER binding assays were 0.4 and 0.75 µM for urolithin A; 20 and 11 µM for urolithin B; 3 and 0.02 for genistein; and 2.3 and 1 for daidzein, respectively; no binding was detected for resveratrol and enterolactone. Urolithins A and B entered into MCF-7 cells and were metabolized to yield mainly urolithin-sulfate derivatives. These results, together with previous studies regarding absorption and metabolism of dietary ellagitannins and ellagic acid in humans, suggest that the gut microflora metabolites urolithins are potential endocrine-disrupting molecules, which could resemble other described “enterophytoestrogens” (microflora-derived metabolites with estrogenic/antiestrogenic activity). Further research is warranted to evaluate the possible role of ellagitannins and ellagic acid as dietary “pro-phytoestrogens”. KEYWORDS: Breast cancer; phytoestrogen; hydroxy-6H-dibenzo[b,d]pyran-6-one derivative; endocrine- disrupting; estrogen receptor INTRODUCTION There is a great concern about the potential of the so-called “endocrine-disrupting chemicals” (EDCs) to either mimic or inhibit endogenous hormones such as estrogens and androgens. Kavlock et al. defined an EDC as an “exogenous agent that interferes with the production, release, transport, metabolism, and binding, action or elimination of natural hormones in the body responsible for the maintenance of homeostasis and the regulation of developmental processes” (1). These compounds are present in the environment and range from natural plant- derived molecules to man-made substances, such as pharma- cological agents and organochlorine pesticides. EDCs may behave physiologically as agonists like endogenous estrogen, oppose estrogen action as an antagonist, or even have mixed agonist properties, behaving as agonists in some tissues and as antagonists in others. This mixed agonist/antagonist effect is characteristic of a new and potentially therapeutically useful class of EDCs, termed “selective estrogen receptor modulators” (SERMs) (2). These compounds are under intense research for their potential to treat estrogen-related conditions such as malignancies and osteoporosis and to alleviate the symptoms associated with menopause. From a dietary point of view, the plant-derived EDCs (“phytoestrogens”) have attracted the attention as “natural” SERMs. Currently, four different classes of phytoestrogens have been dientified: the stilbenes (mainly resveratrol), lignans (converted to enterolactone and enterodiol by gut microflora), coumestans (predominantly coumestrol), and isoflavonoids, which are by far the most studied phytoestrogens (among others, genistein and daidzein, as well as the microflora-derived metabolites such as equol). * Corresponding author (telephone +34-968-396344; fax +34-968- 396213; e-mail jcespin@cebas.csic.es). J. Agric. Food Chem. 2006, 54, 1611-1620 1611 10.1021/jf0527403 CCC: $33.50 © 2006 American Chemical Society Published on Web 02/11/2006