Sulfation of Environmental Estrogens by Cytosolic Human Sulfotransferases

Drug Metabol. Pharmacokin. 17 (3): 221–228 (2002).

Regular Article Sulfation of Environmental Estrogens by Cytosolic Human Sulfotransferases Takahito NISHIYAMA1, Kenichiro OGURA1, Hiroaki NAKANO2, Teppei KAKU1, Eriko TAKAHASHI1, Yasunari OHKUBO1, Koji SEKINE1, Akira HIRATSUKA1, Shigetoshi KADOTA3 and Tadashi WATABE1,3 1Department

of Drug Metabolism and Molecular Toxicology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji-shi, Tokyo, Japan 2Corporate, Scientiˆc and Regulatory AŠairs Division, Tobacco Headquarters, Japan Tobacco Inc., Minato-ku, Tokyo, Japan 3Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, Sugitani, Toyama, Japan

Summary: It is known that in humans taking soy food, the phytoestrogens, daidzein (DZ) and genistein (GS), exist as sulfates and glucuronides in the plasma and are excreted as conjugates in urine. To investigate which human sulfotransferase (SULT) isoforms participate in the sulfation of these phytoestrogens, the four major cytosolic SULTs, SULT1A1, SULT1A3, SULT1E1, and SULT2A1, occurring in the human liver were bacterially expressed as His-tagged proteins and chromatographically puriˆed to homogeneity in the presence of Tween 20 and glycerol as highly e‹cient agents for stabilizing the recombinant enzymes. All the SULTs showed sulfating activity toward both DZ and GS. However, kcat W Km values observed indicated that these phytoestrogens were sulfated predominantly by SULT1A1 and SULT1E1 with Km values of 0.3 and 0.7 mM for GS and 1.9 and 3.4 mM for DZ, respectively. DZ and GS strongly inhibited the sulfation of the endogenous substrate, b-estradiol, by SULT1E1 in a non-competitive manner with Ki values of 14 and 7 mM, respectively, suggesting that these phytoestrogens might aŠect tissue levels of b-estradiol in the human. The phenolic endocrine-disrupting chemicals, bisphenol A (BPA), 4-n-nonylphenol (NP), and 4-t-octylphenol ( t-OP), were used as substrates to investigate the possible participation of human SULTs in their metabolism for excretion. High kcat W Km values were observed for the sulfation of BPA by SULT1A1, NP by SULT1A1 and SULT1E1, and t-OP by SULT1E1 and SULT2A1.

Key words: environmental estrogen; endocrine-disrupting chemicals; human; kinetic parameter; sulfotransferase; phytoestrogen lently modifying DNA. Human liver cytosol contains four major well-characterized cytosolic SULTs: SULT1A1 (previously designated P-PST-1) that sulfates endogenous and exogenous phenols, SULT1A3 (previously M-PST) that sulfates phenolic monoamines, SULT1E1 (previously EST) that sulfates endogenous estrogens, and SULT2A1 (previously DHEA-ST) that sulfates hydroxysteroids and xenobiotic primary and secondary alcohols.1,2) Environmental estrogen-like chemicals, including pesticides (e.g., DDT), phytoestrogens (e.g., genistein (GS)), industrial chemicals (e.g., bisphenol A (BPA)),

Introduction Mammalian cytosolic sulfotransferases (SULTs) play an important role in the inactivation or detoxiˆcation of a variety of hydrophobic endogenous and exogenous substrates, such as phenols, alcohols, N-oxides, and aliphatic and aromatic primary and secondary amines, by their transformation to the strongly anionic hydrophilic products, O- and N-sulfonates, for excretion.1,2) On the contrary, SULTs also catalyze bioactivation of carcinogenic arylhydroxamic acids,3) N-hydroxyarylamines4) and arylmethanols5,6) to reactive O-sulfates cova-

Received; March 12, 2002, Accepted; June 6, 2002 To whom correspondence should be addressed : Prof. Tadashi WATABE, Ph.D., Department of Drug Metabolism and Molecular Toxicology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji-shi, Tokyo 192-0392, Japan, Tel. +81-426-76-4516, Fax. +81-426-76-4517, E-mail: watabet＠ps.toyaku.ac.jp

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and pharmaceuticals (e.g., diethylstilbestrol) are a topic of considerable current interest in view of their reproductive toxicity. Phytoestrogens, such as the soybean iso‰avones, GS and daidzein (DZ), have drawn considerable attention because of their biological properties as estrogen agonists and antagonists. DZ and GS have been reported to have anticarcinogenic properties because of their ability to inhibit protein kinase and DNA topoisomerase activities.7,8) Epidemiological studies suggest that these iso‰avones may have chemopreventing eŠects on breast cancer.9,10) Interestingly, a sulfate of DZ still had binding ability to estrogen receptors and showed biological activities, such as steroid sulfatase inhibition and natural killer-cell activation.11,12) Although sulfates of DZ and GS are detected in the plasma and urine of humans taking soy foods,13,14) little is known of the SULTs that catalyze the sulfation of these substrates apart from the fact that the human SULTs, SULT1A1 and SULT1E1, had activity to sulfate GS.1) However, no kinetic data, especially on kcat W Km, are available on the sulfation of phytoestrogens by human SULTs. BPA, a chemical used in the production of polycarbonate resins, and alkylphenols, such as 4-n-nonylphenol (NP) and 4-t-octylphenol ( t-OP), utilized as components of industrial neutral detergents, are known to exist in the environments,15) including food in the case of BPA,16) and to have a‹nities for estrogen receptors.17) Considerable attention has been given to their estrogenic properties, as with the phytoestrogens, so that they are termed endocrine-disrupting chemicals (EDCs).18) However, little is known of their sulfation by human SULTs apart from the one ˆnding that SULT1A1 had activity toward BPA,19) although no kinetic data are available. In the present study, we provide the kinetic parameters for the sulfation of DZ, GS, BPA, NP, and t-OP by the four major human SULTs, SULT1A1, SULT1A3, SULT1E1, and SULT2A1, to evaluate the possible participation of human SULTs in the biotransformation of phytoestrogens and EDCs. Materials and Methods Materials: Restriction enzymes and EX Taq polymerase were purchased from Takara Shuzo (Kyoto, Japan), and DZ and GS were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). BPA, 4-n-octylphenol (n-OP) and t-OP were purchased from Tokyo Kasei Co. (Tokyo, Japan), and NP was from GL Science (Tokyo, Japan). pET-14b vector and His-bind metal chelation resin were obtained from Novagen (Madison, WI). Unlabeled 3?-phosphoadenosine 5?phosphosulfate (PAPS) was synthesized as reported mmol) previously.20) [2,4,6,7-3H] b-Estradiol (2.96 TBq W

and [35S]PAPS (50.5 GBq W mmol) were purchased from NEN Life Science Products, Inc. (Boston, MA). Radioactive PAPS was puriˆed by chromatography on a DEAE-cellulose column prior to use by a previously reported method.21) A human liver cDNA library was prepared as reported previously.22) Other reagents used were of analytical grade. Construction of expression plasmids for human SULTs: Construction of Escherichia coli-expression plasmids for SULT1A1, SULT1A3, SULT1E1, and SULT2A1 was performed as described previously with slight modiˆcation.23) In brief, each coding region of human SULTs was ampliˆed by PCR from the human XhoI sites cDNA library, subcloned into the NdeI W (SULT1A1, SULT1A3, and SULT1E1) and NdeI site (SULT2A1) of the pET-14b expression vector, and introduced into E. coli BL21 (DE3) pLysS (Novagen, Madison, WI). The subcloned cDNA sequences determined by using an ABI PrismTM 377 DNA sequencer (Perkin-Elmer, Foster, CA) were in agreement with each of the published SULT1A1 (Genbank accession no. X78283), SULT1A3 (Genbank accession no. L19956), SULT1E1 (Genbank accession no. L25275), and SULT2A1 (Genbank accession no. U08025) cDNAs. Bacterial expression and puriˆcation of recombinant human SULTs: Bacterial expression and puriˆcation of His-tagged recombinant human SULTs by one-step column chromatography were performed as described previously23) with some modiˆcations: a bacterial lysate mL) containing 0.025z (w W v) Tween 20 (25 mg protein W and 10z (w W v) glycerol was loaded onto a His-bind metal chelation resin column (1×5 cm) pre-equilibrated with 20 mM Tris-HCl buŠer (pH 8.0) containing 5 mM v) imidazole, 500 mM NaCl, 1 mM PMSF, 0.025z (w W v) glycerol (buŠer I). After Tween 20, and 10z (w W washing the column with 60 mL of the same buŠer, the recombinant protein was eluted with a step-wise gradient of 15 mL each of 100, 200, 400, and 1000 mM imidazole in buŠer I. Fractions containing the homogeneous enzyme protein, appearing as a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were pooled as described previously.23) The puriˆed enzyme solution was desalted on a PD-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden) pre-equilibrated with 50 mM sodium phosphate buŠer (pH 6.8) containing 8 mM dithiothreitol, v) Tween 20, and 10z (w W v) glycerol. The 0.025z (w W protein concentration was measured by the method of Bradford24) using BSA as a standard and stored at „809 C after the addition of BSA (10 mg W mL). Enzyme assay: Sulfating activities of puriˆed human SULTs toward BPA, dehydroepiandrosterone, dopamine, DZ, GS, 4-nitrophenol, NP, n-OP and t-OP were determined by the method of Sekura et al.21) with

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some modiˆcations. The reaction mixture containing the substrate dissolved in DMSO (2.5 mL), 50 mM sodium phosphate buŠer (pH 6.8 for SULT1A1 and SULT1A3) or 50 mM Tris-HCl buŠer (pH 7.4 for SULT1E1 and SULT2A1), 7 mM MgCl2, 8 mM mL), [35S]PAPS (10 mM, dithiothreitol, BSA (625 mg W 3.7 GBq W mmol), and enzyme (6–300 ng) in a ˆnal volume of 50 mL, was incubated for 10 min at 379 C. After a 2-min preincubation, the reaction was started by the addition of the substrate and terminated by placement of the reaction vessel in an ice bath. An aliquot (5 mL) of the reaction mixture was applied to a polyethyleneimine cellulose thin layer plate (MarchereyNagel GmbH & Co. KG, Dueren, Germany) and devev). loped with t-butanol:ethyl acetate:water (8:6:4, v W Under the TLC condition, sulfate esters of BPA, DZ, GS, NP, n-OP and t-OP migrated at Rf values of 0.8–0.9, whereas unreacted radioactive PAPS and inorganic sulfates remained at the origin. The radioactivity of the sulfate ester was determined by radioluminography with a BAS 2000 bioimaging analyzer (Fuji Photo Film, Tokyo, Japan). Sulfating activity of SULT1E1 toward b-estradiol was determined as reported previously25) using [2,4,6,7-3H] b-estradiol as a substrate. Brie‰y, after incubation, the reaction mixture was made alkaline with 0.5 M Tris-HCl buŠer, pH 8.7 and repeatedly extracted with chloroform to remove the unreacted radioactive substrate. The radioactive bestradiol sulfate in the residual aqueous phase was determined by liquid scintillation counting. The inhibition constant ( Ki ) in b-estradiol sulfation with DZ and GS was determined using a Dixon plot obtained in the zeroorder kinetics region of the enzyme reactions. For calculation of kcat values, molecular weights of the His-tagged SULT proteins used were 36359 for SULT1A1, 36358 for SULT1A3, 37288 for SULT1E1, and 35927 for SULT2A1. Data were expressed as means of at least three experiments. Results Kinetic properties of puriˆed recombinant human SULTs: Four major SULT1A1, SULT1A3, SULT1E1, and SULT2A1 occurring in the human liver were bacterially expressed as His-tagged proteins and puriˆed from bacterial cytosol on a Ni-binding resin column in the presence of Tween 20 and glycerol, which were highly eŠective to prevent the recombinant enzymes from irreversible inactivation by coagulation. Using these recombinant human SULTs obtained in stable form, we determined kinetic parameters for the sulfation of the typical substrates, 4-nitrophenol by SULT1A1, dopamine by SULT1A3, b-estradiol by SULT1E1, and dehydroepiandrosterone by SULT2A1 (Table 1). The SULTs chromatographically puriˆed in the absence of Tween 20 and glycerol exhibited very low

Table 1. Kinetic properties in the sulfation of typical substrates by puriˆed recombinant human SULTs SULT

Substrate

Kma) ( mM)

kcatb) (min„1)

kcat W Km (min„1・ mM„1)

SULT1A1 SULT1A3 SULT1E1 SULT2A1

4-Nitrophenol Dopamine b-estradiol Dehydroepiandrosterone

0.2 1.5 0.02 2.2

13.8 4.9 1.4 12.6

65905 3212 71000 5741

Enzyme activities were determined as described in Materials and Methods. Parameters were obtained from Lineweaver-Burk plots and expressed as mean values of at least three experiments. a) Kinetic parameters for sulfations of the typical substrates by SULT1A1, SULT1A3, SULT1E1, and SULT2A1 were obtained by double-reciprocal plots of reaction rates vs substrate concentrations ranging from 0.1 to 10 mM for 4-nitrophenol, 0.25 to 4 mM for dopamine, 2.5 to 60 nM for b-estradiol, and 0.25 to 10 mM for dehydroepiandrosterone, respectively, in the zero-order kinetics region. To calculate the kinetic parameters for the sulfation by SULT1A1 and SULT2A1, reaction rates at the substrate concentrations lower than 2 mM for 4-nitrophenol and 3 mM for dehydroepiandrosterone were used as these substrates inhibited enzymatic sulfation at concentrations higher than these substrate concentrations. b) Molecular weights of SULTs are calculated values for molecularly cloned His-tagged peptides: 36359 for SULT1A1, 36358 for SULT1A3, 37288 for SULT1E1, and 35927 for SULT2A1.

kcat values compared with those shown in Table 1, although only a little diŠerence was observed in Km. For instance, kcat for SULT1A1 puriˆed in the absence of these stabilizing agents during chromatography varied 40th to 1 W 100th of that in Table 1. A reproducifrom 1 W ble kcat value was observed for each enzyme puriˆed in the presence of Tween 20 and glycerol, but not in their absence. In addition, enzyme activity was not enhanced when Tween 20 and glycerol were added to the SULTs puriˆed from bacterial cytosol in their absence, strongly suggesting that they had no stimulating eŠect on the enzyme activity. Sulfation of the phytoestrogens, DZ and GS, by puriˆed recombinant human SULTs: A kinetic study using DZ and GS as substrates indicated that all the aforementioned four major recombinant human SULTs had sulfating activity toward the phytoestrogens (Table 2). However, SULT1A1 and SULT1E1 exhibited much Km, than did the other higher catalytic e‹ciency, kcat W Km two, SULT1A3 and SULT2A1. The observed kcat W indicated that GS was a 12- and 8-fold better substrate than DZ for SULT1A1 and SULT1E1, respectively. The large diŠerence in the catalytic e‹ciency of the SULTs was mainly due to Km rather than kcat; Km for sulfation by SULT1A1 and SULT1E1 was 6- and 5-fold higher, respectively, for DZ than for GS. A much higher Km was observed for sulfation of the phytoestrogens by SULT1A3 and SULT2A1 than by SULT1A1 and SULT1E1.

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Inhibitory eŠects of DZ and GS on b-estradiol sulfation by SULT1E1: A study using SULT1E1 indicated Table 2. Michaelis-Menten parameters for the sulfation of DZ and GS by puriˆed recombinant human SULTs DZa) SULT

Kmb) ( mM)

kcatc) (min„1)

SULT1A1 SULT1A3 SULT1E1 SULT2A1

1.7 33.6 3.4 167.3

1.1 0.4 4.0 0.2

GSa) kcat W Km Kmb) (min„1・ ( mM) mM„1)

647.1 11.9 1176.5 1.2

0.3 34.0 0.7 41.0

kcatc) (min„1)

kcat W Km (min„1・ mM„1)

2.4 0.7 6.4 0.1

8000.0 20.6 9142.9 2.4

Enzyme activities were determined at substrate concentrations ranging 0.03 to 1000 mM as described in Materials and Methods. Parameters were obtained from Lineweaver-Burk plots and expressed as mean values of at least three experiments. a) DZ: daidzein; GS: genistein. b) Km and Vmax used for calculation of kcat were obtained by extrapolation of reaction rates vs substrate concentrations lower than 2 mM for DZ and 0.5 mM for GS as the phytoestrogens inhibited enzymatic sulfation at concentrations higher than these substrate concentrations. c) See the legend under Table 1 for molecular weights of SULTs.

Table 3.

that DZ and GS inhibited the sulfation of b-estradiol in a non-competitive manner with ki values of 14 and 7 mM estimated by dixon plots, respectively (Fig. 1). Sulfation of endocrine-disrupting chemicals by puriˆed recombinant human SULTs: SULT1A1, SULT1E1, and SULT2A1 showed sulfating activity toward the synthetic estrogenic phenols, BPA, NP, and t-OP (Table 3). However, SULT1A3 had no detectable activity toward BPA and t-OP, but showed only a little activity toward NP, which was also a very poor substrate for SULT2A1 compared with the other two EDCs. NP with a straight C9 side chain at the 4-position of phenol was sulfated by SULT1A1 and SULT1E1 Km among the with the lowest Km and the highest kcat W three EDCs. On the contrary, t-OP with a highly branched C8 side chain at the 4-position of phenol was sulfated by SULT1E1 and SULT2A1 with a relatively low Km. The sulfation of t-OP by SULT1A1 proceeded with a high Km compared with that by SULT1E1 and SULT2A1. The SULT1A1-mediated sulfation of n-OP with a straight C8 chain at the 4-position of phenol proceeded at 1 W 27th of the Km for t-OP and a 5-fold higher kcat

Michaelis-Menten parameters for the sulfation of BPA, NP, and t-OP by puriˆed recombinant human SULTs BPAa)

NP

t-OP

SULT

Km ( mM)

kcat (min„1 )

kcat W Km (min„1・mM„1)

Km ( mM)

kcat (min„1 )

kcat W Km (min„1・mM„1)

Km ( mM)

kcat (min„1 )

kcat W Km (min„1・mM„1)

SULT1A1 SULT1A3 SULT1E1 SULT2A1

4.7

2.7 N.C.b) 2.5 0.2

574.4

2.8 188.4 2.5 71.5

6.3 0.7 5.3 0.05

2250.0 3.7 2120.0 0.7

27.0

1.9 N.C.b) 3.7 1.1

70.4

43.0 4.3

58.1 46.5

7.8 5.1

474.3 215.7

Enzyme activities were determined as described in Materials and Methods. The concentrations of BPA, NP, and t-OP were varied between 0.5 and 600 mM. Parameters were obtained from Lineweaver-Burk plots and expressed as mean values of at least three experiments. a) BPA (bisphenol A: 4,4?-isopropylidenediphenol), NP (4-n-nonylphenol), t-OP (4-t-octylphenol: [4-(1,1,3,3-tetramethylbutyl)phenol]). b) N.C.: not conjugated (less than 0.02 min„1).

Fig. 1. Dixon plots of inhibition of SULT1E1-dependent b-estradiol sulfation with DZ and GS. Puriˆed SULT1E1 (1 ng W mL) was incubated with 10 (), 20 (#), or 40 () nM [3H] b-estradiol as a substrate at 379C for 10 min in the presence of various concentrations of DZ (0 to 20 mM) or GS (0 to 20 mM). Data are arithmetic mean values of at least three experiments.

Sulfation of Environmental Estrogens

Fig. 2.

225

Chemical structures of phytoestrogens and endocrine-disrupting chemicals (EDCs).

than that of t-OP. Discussion As reported previously by us on the column chromatographic puriˆcation of rat liver cytosolic SULTs,26) the neutral detergent, Tween 20, added to the mobile phase was also very eŠective for the puriˆcation of recombinant human SULTs to prevent from irreversible denaturation with signiˆcant loss of enzyme activity. A high concentration of glycerol was also eŠective for this purpose. In the present study, a combination of Tween 20 with the glycerol was found to be more eŠective than the single use of these stabilizing agents. The catalytic e‹ciency, kcat W Km, of our recombinant human SULTs puriˆed in the presence of Tween 20 and glycerol (Table 1) were 60-, 8-, 74-, and 18-fold higher for SULT1A1, SULT1A3, SULT1E1, and SULT2A1, respectively, than those reported by Fujita et al.27) They used Tween 20 and glycerol for the stabilization of recombinant SULTs after being puriˆed by chromatography as Lewis et al.23) had done so for recombinant SULT1A1 using glycerol alone. In this study, the phytoestrogens, DZ and GS, were strongly suggested to be predominantly sulfated by SULT1A1 and SULT1E1 in the human (Table 2). Our Km, SULT1E1 was data indicated that based on kcat W more active toward DZ and GS than SULT1A1, whereas Falany1) had reported that GS was a better substrate for SULT1A1 than for SULT1E1 based on apparent speciˆc activity of their recombinant human SULTs. It is of interest that these phytoestrogens are good substrates for SULT1E1 playing an important role in the sulfation of estrogens for excretion in vivo.1,2) From the viewpoint of a structure-activity relationship, it is of interest that GS was demonstrated to have much higher a‹nity, more than 13 times, for human

estrogen receptors than DZ28) and that GS was a better substrate for SULT1E1 than DZ (Table 2). GS is a 5hydroxy-DZ and the 5-hydroxyl group forms a strong intramolecular hydrogen bond with the 4-carbonyl group (Fig. 2). Therefore, the 5-hydroxyl group was demonstrated to be resistant to acetylation with acetic anhydride in pyridine at room temperature, whereas the 7- and 4?-hydroxyl groups are readily acetylated under this condition.29) The strong hydrogen bonding with the formation of an additional 6-membered ring to the iso‰avone may extend its planar molecular size. That may be why GS shows higher a‹nity for SULT1E1 and estrogen receptors than does DZ. Although the diŠerence in km was extremely marked between sulfation of b-estradiol (0.02 mM) and GS (0.7 mM) or DZ (3.4 mM) by SULT1E1 (Table 1 and 2), the observed inhibition constants, Ki, 7 mM for GS and 14 mM for DZ, suggest that the enzyme may be strongly inhibited by the phytoestrogens at tissue concentrations around Ki. Sulfation of b-estradiol by SULT1E1 was demonstrated by dixon plots to be inhibited with GS and DZ in a non-competitive manner. However, these phytoestrogens may act as competitive inhibitors on the b-estradiol sulfation at concentrations lower than 0.5 and 2 mM for GS and DZ, respectively, which were sulfated as good substrates by SULT1E1 without exhibiting the substrate inhibition. Actually, Harrison et al.30) recently reported that in pregnant rhesus monkeys, the long-term daily oral kg body administration of a daily dose of GS (8 mg W weight) resulted in elevated serum concentrations of bestradiol to levels 1.5- to 2.0-fold higher than in the controls and also that the serum b-estradiol levels in fetuses delivered by cesarean section were as high as those in the GS-treated mothers. However, studies were not done to determine the mechanism of such an elevation of serum

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b-estradiol levels. Sulfation of b-estradiol by human SULT1E1 has been demonstrated by Kester et al.31) to be inhibited noncompetitively with extremely low concentrations of hydroxypolychlorinated biphenyls through an allosteric site of the enzyme. Zhang et al.32) also suggested the existence of at least two inhibition sites, allosteric and substrate-binding (active) sites, in the SULT1E1 protein. Soybeans are one of the most representative sources for DZ and GS, which occur as glycosides in the beans. The glycosides readily release the iso‰avones by micro‰ora in the intestines. The iso‰avones show opposite activities as estrogen agonists and antagonists, which depend on the concentration of phytoestrogens.7,10) Focusing on the estrogen antagonist activity of DZ and GS, a soy extract packed in capsules is being widely distributed on the international market for the chemoprevention of breast cancer. However, it should be noted that 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 the aforementioned pregnant rhesus monkeys given GS.30) In sheep33) and rats,34) phytoestrogens have been demonstrated to cause reproductive problems. Recently, Doerge et al.35) reported a kinetic study of DZ and GS sulfation using commercially available recombinant human SULTs. They stated that microsomes were used as a source of recombinant human SULTs expressed in Sf9 cells transformed by vaculoviruses which separately contained cDNAs coding for SULT1A1, SULT1A3, SULT1E1, and SULT2A1. Recombinant and native SULTs do occur in cytosol in cells. In addition, they stated that none of the recombinant human SULTs sulfated DZ and also that GS was sulfated by SULT1A1 and SULT1E1 at Km values approximately 1000- and 1500- fold higher, respectively, than observed in our study which used freshly prepared and puriˆed recombinant human SULTs. As to EDCs, BPA was sulfated by SULT1A1, NP by SULT1A1 and SULT1E1, and t-OP by SULT1E1 and Km (Table 3). There SULT2A1 at a relatively high kcat W was a marked diŠerence in Km between the sulfation of t-OP and NP or n-OP by SULT1A1. That might be attributable to the highly branched bulky side chain of tOP and the straightly extended side chain of NP and nOP because SULT1A1 has been recognized to catalyze the sulfation of planar simple phenols such as 4nitrophenol and 1- and 2-naphthols as good substrates.1) Despite its bulky structure, the symmetric and bifunctional phenol, BPA, was sulfated by SULT1A1 with a relatively high kcat W Km compared with those by SULT1E1 and SULT2A1. Sulfation of various alkylphenols including NP in human platelet cytosol has been reported by Harris et al. (36). They suggested short chain 4-n-alkylphenols

(Cº8) were substrates for SULT1A1 W 2, and long chain 4-n-substituted alkylphenols (CÀ8) were poor substrates and act as inhibitors of SULT1A1 W 2 based on experiments using inhibitors speciˆc for SULT isoforms. They also demonstrated that NP was a partial mixed inhibitor for a low a‹nity activity of b-estradiol sulfation in platelet cytosol. However, they provided no direct evidence for the SULT isoforms involved in the sulfation of alkylphenols and b-estradiol because they used only cytosol from human platelets as an enzyme source. Very recently, Suiko et al.37) reported the sulfation of the EDCs, BPA, NP, and n-OP, by recombinant human SULTs puriˆed in the absence of stabilizing agents such as Tween 20 and glycerol. However, the apparent speciˆc activities of their SULTs toward the EDCs were extremely low: one to three orders of magnitude lower at a single substrate concentration of 50 mM than our data determined at the same substrate concentration. Their SULTs were likely to have been denatured with loss of most activity during puriˆcation because reported apparent speciˆc activities toward the typical substrates such as 4-nitrophenol and dopamine for these enzymes were also very low.38) DZ and GS were reported to be excreted as monoand di-sulfates, mono- and di-glucuronides, and sulfoglucuronides in human urine.14) No enzymatic study has been made on the production of these di-conjugates. A study on the di-conjugation reaction of the phytoestrogens by human SULTs and UDP-glucuronosyltransferases is now in progress in our laboratory. References 1) 2)

3)

4)

5)

6)

7)

Falany, C. N.: Enzymology of human cytosolic sulfotransferases. FASEB J., 11: 206–216 (1997). Nagata, K. and Yamazoe, Y.: Pharmacogenetics of sulfotransferase. Annu. Rev. Pharmacol. Toxicol., 40: 159–176 (2000). DeBaun, J. R., Rowley, J. Y., Miller, E. C. and Miller, J. A.: Sulfotransferase activation of N-hydroxy-2acetylamino‰uorene in rodent livers susceptible and resistant to this carcinogen. Proc. Soc. Exp. Biol. Med., 129: 268–273 (1968). Kadlubar, F. F,, Miller, J. A. and Miller, E. C.: Hepatic metabolism of N-hydroxy-N-methyl-4-aminoazobenzene and other N-hydroxy arylamines to reactive sulfuric acid esters. Cancer Res., 36: 2350–2359 (1976). Watabe, T., Ishizuka, T., Isobe, M. and Ozawa, N.: A 7-hydroxymethyl sulfate ester as an active metabolite of 7,12-dimethylbenz[a]anthracene. Science, 215: 403–405 (1982). Okuda, H., Hiratsuka, A., Nojima, H. and Watabe, T.: A hydroxymethyl sulphate ester as an active metabolite of the carcinogen, 5-hydroxymethylchrysene. Biochem. Pharmacol., 35: 535–538 (1986). Barnes, S. and Peterson, T. G.: Biochemical targets of the iso‰avone genistein in tumor cell lines. Proc. Soc. Exp. Biol. Med., 208: 103–108 (1995).

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8)

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