PHENOL SULFOTRANSFERASE, ST1A3, AS THE MAIN ENZYME CATALYZING SULFATION OF TROGLITAZONE IN HUMAN LIVER WATARU HONMA, MIKI SHIMADA, HIRONOBU SASANO, SHOGO OZAWA, MASAAKI MIYATA, KIYOSHI NAGATA, TOSHIHIKO IKEDA, AND YASUSHI YAMAZOE Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan (W.H., M.S., M.M., K.N., and Y.Y.); Department of Anatomic Pathology, School of Medicine, Tohoku University, Sendai, Japan (H.S.); Division of Pharmacology, National Institute of Health Sciences, Tokyo, Japan (S.O.); and Pharmacokinetics and Drug Delivery Research Laboratories, Sankyo Co., Ltd., Tokyo, Japan (T.I.) (Received November 2, 2001; accepted May 15, 2002) This article is available online at http://dmd.aspetjournals.org ABSTRACT: Since sulfation is the main metabolic pathway of troglitazone, accounting for about 70% of the metabolites detected in human plasma, we have aimed to identify human cytosolic sulfotrans- ferases catalyzing the sulfation of troglitazone and to examine a possible role of the sulfation in the cytotoxicity observed in cell lines of human origin (HepG2 and Hep3B). Experiments using the recombinant sulfotransferases and human liver cytosols indicated that phenol sulfotransferase (ST1A3) and estrogen sulfotrans- ferase (ST1E4) were the sulfotransferases most active toward tro- glitazone. Immunoblot analyses indicated that hepatic content of ST1A3 is about 13 times higher than that of ST1E4, suggesting that ST1A3 is mainly responsible for the sulfation of troglitazone in the liver. Lactate dehydrogenase (LDH) leakage was elicited by trogli- tazone in a concentration-dependent manner in the hepatoma cells. The troglitazone metabolites (the sulfate, glucuronide, and quinone forms) caused negligible LDH leakage. These findings suggest that accumulation of unmetabolized troglitazone causes the cytotoxicity in the hepatoma cells and may be responsible for toxicity in human liver. Troglitazone (Rezulin, Warner-Lambert Co. or Noscal, Sankyo Co., Ltd.) has been used as an oral antidiabetic drug for the treatment of non-insulin-dependent diabetes mellitus. It lowers blood glucose levels through increasing glucose uptake at skeletal muscles, decreasing hepatic glucose production and increasing sensitivity to insulin (Ciaraldi et al., 1990; Fujiwara et al., 1995, 1998). A rare but serious idiosyncratic hepatotoxicity due to the troglitazone-treatment has been reported (Watkins and Whitcomb, 1998), leading to the withdrawal of this drug from the market and leaving the mechanism of toxicity obscure. The sulfate and qui- none forms of troglitazone are the major metabolites, whereas the glucuronide form is a minor metabolite in humans. Troglitazone sulfate accounted for about 70% of the metabolites detected in human plasma (Shibata et al., 1993; Loi et al., 1997). Thus, sulfation is considered the major pathway in troglitazone metabo- lism, determining the exposure to this drug. Little information on enzymatic sulfation of troglitazone has, however, been reported. Despite its withdrawal, study of the metabolism would contribute to understanding the mechanism of toxicity caused by troglitazone. Cytosolic sulfotransferases (STs or SULTs 1,2 ) catalyze sulfation of various endogenous and exogenous chemicals (De Meio, 1975; Ja- koby et al., 1980; Yamazoe and Kato, 1995; Nagata and Yamazoe, 2000). The reaction involves the transfer of a sulfonate group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the substrate. STs are known to constitute a gene superfamily, which contains at least five subfamilies (ST1 to ST5) in mammals based on the similarities of their deduced amino acid sequences. There are differences in the substrate specificities and expression profiles among ST forms (Yamazoe et al., 1994; Weinshilboum et al., 1997; Dooley et al., 2000; Nagata and Yamazoe, 2000). In the present study, we have aimed to identify the human cytosolic sulfotransferases catalyzing the sulfation of troglitazone and to examine a possible role of sulfation in the toxicity of troglitazone using hepatoma cell lines of human origin. Experimental Procedures Materials. Troglitazone (purity 99%), its sulfate (98%) and glucuronide (98%) conjugates and its quinone-type metabolite [()-5-(4-(2-hydroxy-2- methyl-4-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)butoxy)benzyl)-2,4-thiazoli- dine-dion, purity 98%] were provided by Sankyo Co. Ltd. (Tokyo, Japan). This study was supported in part by grant-in-aids from the Ministry of Educa- tion, Science and Culture and the Ministry of Health and Welfare, Japan; the Japan Health Sciences Foundation, Smoking Research Foundation, Japan; and Human & Animal Bridge Discussion Group, Japan. Address correspondence to: Yasushi Yamazoe, Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki-Aoba, Aoba-ku, Sendai, 980-8578, Japan. E-mail: yamazoe@mail.cc.tohoku.ac.jp 1 Abbreviations used are: ST or SULT, cytosolic sulfotransferase; PAPS, 3'- phosphoadenosine-5'-phosphosulfate; His-ST, recombinant ST protein that has additional amino acid residues at the N-terminal; His-ST, fused portion removed from His-ST by digestion with enterokinase; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; HPLC, high-performance liquid chromatog- raphy; LDH, lactate dehydrogenase. 2 STs or SULTs are as follows: ST1A3, ST1A5, ST1B2, ST1C2, ST1E4, and ST2A3 correspond to SULT1A1, SULT1A3, SULT1B1, SULT1C2, SULT1E1 and SULT2A1, respectively. 0090-9556/02/3008-944–949$7.00 DRUG METABOLISM AND DISPOSITION Vol. 30, No. 8 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 633/1001304 DMD 30:944–949, 2002 Printed in U.S.A. 944 at ASPET Journals on April 5, 2017 dmd.aspetjournals.org Downloaded from