Regioselective monosulfation and disulfation of the phytoestrogens daidzein and genistein

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1Corporate,
Summary: Regioselective sulfation of the phytoestrogens daidzein (DZ, 7,4?-dihydroxyiso‰avone) and genistein (GS, 5,7,4?-trihydroxyiso‰avone) was investigated using human liver cytosol and puriˆed recombinant human sulfotransferase (SULT) isoforms, SULT1A1, SULT1A3, SULT2A1, and SULT1E1. 7-Position-preferential sulfation of DZ and GS was observed in human hepatic cytosols from 3 male and 3 female subjects. Average ratios for 7- to 4?-sulfate formation were 4.5:1 from DZ and 8.4:1 from GS in these human liver cytosols. Apparent Km values for the 7- and 4?-sulfation of DZ and GS by these cytosols were similar and in a range from 0.46 to 0.66 mM. All recombinant human SULTs had activity for 7- and 4?-sulfation of these phytoestrogens except for 7-sulfating activity of SULT1A3. Km, for 7- and 4?-sulfation of SULT1A1 and SULT1E1 exhibited much higher catalytic e‹ciency, kcat W these substrates than did the other two, SULT1A3 and SULT2A1. SULT1A1 showed Km values of 0.47 and 0.52 mM for the mono-sulfation of DZ and GS, respectively, which were very similar to those of Km indicated that SULT1A1 catalyzed 7-sulfation of DZ and GS at human cytosol. The observed kcat W rates 4.4- and 8.8-fold higher, respectively, than such 4?-sulfation. However, with SULT1E1, catalytic e‹ciency was very similar for the sulfation of both positions. These data strongly suggest that SULT1A1 plays a major role in monosulfation of the phytoestrogens and determines the regioselectivity of sulfation in human hepatic cytosol. A kinetic study for 7,4?-disulfate formation of DZ and GS from their 7- and 4?-monosulfates indicated that SULT1E1 most e‹ciently catalyzed both reactions among human SULTs.
Scientiˆc and Regulatory AŠairs Division, Tobacco Headquarters, Japan Tobacco Inc., Tokyo, Japan 2Department of Drug Metabolism and Molecular Toxicology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan 3Division of GI Oncology, National Cancer Center Hospital, Tokyo, Japan 4Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan
Drug Metab. Pharmacokin. 19 (3): 216–226 (2004).
Regular Article
Regioselective Monosulfation and Disulfation of the Phytoestrogens Daidzein and Genistein by Human Liver Sulfotransferases
Hiroaki NAKANO1, Kenichiro OGURA2, Eriko TAKAHASHI2, Tomokazu HARADA2, Takahito NISHIYAMA2, Kei MURO3, Akira HIRATSUKA2, Shigetoshi KADOTA4 and Tadashi WATABE2,4
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Regioselective Sulfation of Daidzein and Genistein
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phytoestrogens actually serve as chemopreventive agents in humans. DZ and GS are known to show opposite activities as estrogen agonists and antagonists, which from in vitro studies were shown to be concentration dependent.10) Focusing on the estrogen antagonist activity of these iso‰avones, many kinds of soy extract powders, capsules and tablets are being widely distributed on the international market as supplements for the chemoprevention of breast cancer. Although many putative beneˆcial eŠects of phytoestrogens were demonstrated, some studies showed their potential toxicity. The daily intake of soy extract containing large amounts of iso‰avone glycosides might have elevated the fetal b-estradiol level as observed in the fetuses of pregnant Rhesus monkeys given GS.11) Cassidy et al.12) found that ingesting 60 g of soy protein (equivalent to 45 mg iso‰avones) daily for 1 month was su‹cient to disrupt the menstrual cycle in premenopausal women. In sheep13) and rats,14) phytoestrogens have been demonstrated to cause reproductive problems. Although DZ and GS can be detected in the plasma and urine of humans who consume soy foods, the majority of these iso‰avones circulate and are excreted into urine as mono- and di-sulfates, mono- and diglucuronides, and sulfoglucuronides.15) DZ and GS aglycons are associated with biological activity; however, sulfate conjugates of DZ were demonstrated to still have binding ability to estrogen receptors and showed biological activity, such as sterol sulfatase inhibition and natural killer-cell activation.16,17) GS glucuronides may also have weak estrogenic activity and can activate human natural killer cells in vitro.17) It is, therefore, suggested that the conjugation reactions of these phytoestrogens not only involve deactivation for excretion, but also may involve metabolic activation. It is also possible that the sulfate and glucuronide conjugates of iso‰avones can be a source of biologically active free aglycons at target tissues after enzymatic hydrolysis by sulfatase or b-glucuronidase. It is known that human liver cytosol contains four major well-characterized sulfotransferases (SULTs): SULT1A1 that sulfates endogenous and exogenous phenols, SULT1A3 that sulfates phenolic monoamines, SULT1E1 that sulfates endogenous estrogens, and SULT2A1 that sulfates hydroxysteroids and xenobiotic primary and secondary alcohols.18,19) We have recently reported kinetic studies showing sulfate conjugation of DZ and GS using bacterially expressed human SULTs, SULT1A1, SULT1A3, SULT1E1, and SULT2A1.20) Our data showed that human SULT1A1 and SULT1E1 predominantly catalyzed the sulfation of DZ and GS. However, although DZ and GS have two and three phenolic hydroxyl groups, respectively, which can be sulfonated, the position where sulfation takes place,