Short Communication THE DIETARY POLYPHENOL ELLAGIC ACID IS A POTENT INHIBITOR OF hOAT1

Ellagic acid (EA), a polyphenol present in berries, has been demonstrated to be preventive of esophageal and colon cancer in animals. Here, we have studied the ability of organic anion transporters (OATs) and organic anion-transporting polypeptides (OATPs) to transport EA. The accumulation of radiolabeled [C]EA, [H]p-aminohippuric acid (PAH), [C]glutarate, [H]estrone sulfate, [H]ochratoxin A, and [H]taurocholic acid inhibitor(s) was tested in OATand OATP-expressing oocytes. Oocytes expressing human (h)OAT1, rat (r)Oat1, and hOAT4 accumulated 6.5-, 7.1-, and 8.9-fold more EA, respectively, than did water-injected oocytes. This accumulation was prevented by the prototype OAT inhibitors bromosulfophthalein and probenecid. rOatp1, mouse (m)Oat2, hOAT3, and mOat5 showed no EA transport. The uptake of the prototype OAT substrate PAH in hOAT1-expressing oocytes was dose dependently and potently inhibited by EA with an IC50 of 207 nM. In conclusion, we have demonstrated that the OAT family members hOAT1, rOat1, and hOAT4 mediate transport of EA, with a very high affinity for hOAT1. Organic anion transporters (OATs) play a critical role in the distribution and elimination of a diverse array of exogenous and endogenous compounds. The substrates of OATs are small organic anions at physiologic pH and include a multitude of clinically used therapeutics such as angiotensin-converting enzyme inhibitors, -lactam antibiotics, and nonsteroidal anti-inflammatory drugs. Certain drugs that are eliminated from the body mainly by the kidneys through the OATs can compete with other OAT substrates for transport. This competition for transport can cause retention of certain drugs, leading to longer plasma half-lives (Burckhardt and Burckhardt, 2003). Historically, the interaction was utilized to maintain penicillin plasma levels with the use of probenecid. Plant polyphenols are the focus of much research for the ability to affect adverse human biological disease states. One of these polyphenols, ellagic acid (EA), found naturally in our diet (Lei et al., 2001), has been demonstrated to be a cancer-preventive agent for esophageal cancer in animal models (Stoner and Gupta, 2001). EA is a small organic anion at physiologic pH (Priyadarsini et al., 2002), and its accumulation in human intestinal Caco-2 cells was demonstrated to have OAT-like properties (A. C Whitley, D. H. Sweet, and T. Walle, manuscript submitted for publication). Studies in mice have noted that the kidney is the primary route of elimination for EA equivalents (Teel and Martin, 1988), and it is also the tissue in which OATs have particular importance. Therefore, the present study was focused on determining the ability of the OATs to transport EA and whether EA could influence OAT transport. Materials and Methods Materials. [C]Ellagic acid was synthesized with a specific activity of 20 mCi/mmol at the Ohio State University Comprehensive Cancer Center (Zeng et al., 1991) and was a kind gift from Dr. Gary Stoner, Ohio State. [H]Taurocholic acid (3.5 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). [C]Glutarate (55 mCi/mmol) was purchased from MP Biomedicals (Irvine, CA). [H]p-Aminohippuric acid (20 Ci/mmol), [H]estrone sulfate (57 Ci/mmol), and [H]ochratoxin A (15 Ci/mmol) were purchased from American Radiolabeled Chemicals (St. Louis, MO). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Xenopus laevis Oocyte Uptake Assay. X. laevis oocytes were obtained as described previously (Youngblood and Sweet, 2004). Substrate uptake assays were performed 3 days after injection with 20 ng of capped cRNA (hOAT1, rOat1, mOat2, hOAT3, hOAT4, mOat5, or rOatp1), as previously described (Cihlar et al., 1999). Oocytes were randomly divided into experimental groups of 10 and incubated for 60 min (Sweet et al., 1997) in 1 ml of oocyte Ringer medium containing 20 M [C]EA in all types of oocytes, 10 M [H]paminohippurate (PAH) in hOAT1 and rOat1 oocytes (Pritchard and Miller, 1993), 1 M [H]estrone sulfate in hOAT3 oocytes (Cha et al., 2001), 36.4 M [C]glutarate in mOat2 oocytes (Kobayashi et al., 2002), 1 M [H]ochratoxin A and 1 M [H]estrone sulfate in hOAT4 oocytes (Cha et al., 2000), 1 M [H]ochratoxin A in mOat5 oocytes (Youngblood and Sweet, 2004), and 300 nM [H]taurocholate in rOatp1 oocytes (Li et al., 1998). The uptake inhibitor used was 1 mM probenecid (Cihlar et al., 1999) or 500 M bromosulfophthalein (BSP) (A. C Whitley, D. H. Sweet, and T. Walle, manuscript submitted for publication). Other inhibitors used were 0.05 to 35 M EA, 5 to 200 M indoxyl sulfate, or 50 to 900 M sodium salicylate (Khamdang et al., 2002; Enomoto et al., 2003). Oocytes were rapidly rinsed four times with ice-cold oocyte Ringer medium, and after digestion with sodium hydroxide, individual oocyte radioactivity was measured by liquid scintillation spectroscopy with external quench correction. Each experiment was repeated in three different animals. Water-injected oocytes were included as negative controls in each experiment. Statistics. Data are expressed as means S.E. Statistical differences were determined using analysis of variance followed by Dunnett’s multiple comparison test. Differences were considered significant when P 0.05. This work was supported by National Institutes of Health Grant GM55561 and GEO Centers/Department of Defense Grant GC-3532-03-42153CM. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.105.004275. ABBREVIATIONS: OAT, organic anion transporter; EA, ellagic acid; OATP, organic anion-transporting polypeptide; PAH, p-aminohippuric acid; BSP, bromosulfophthalein; OA, ochratoxin A; AZT, 3 -azido-2 ,3 -dideoxythymidine; prefixes h, r, and m denote human, rat, and mouse transporters. 0090-9556/05/3308-1097–1100$20.00 DRUG METABOLISM AND DISPOSITION Vol. 33, No. 8 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 4275/3041680 DMD 33:1097–1100, 2005 Printed in U.S.A. 1097 at A PE T Jornals on July 7, 2017 dm d.aspurnals.org D ow nladed from