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Drug Metabolism and Disposition Fast Forward
First published on September 15, 2004; DOI: 10.1124/dmd.104.000919

0090-9556/04/3212-1501-1508$20.00
DMD 32:1501-1508, 2004

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INTERACTIONS OF THE STEREOISOMERS OF {alpha}-HYDROXYTAMOXIFEN WITH HUMAN HYDROXYSTEROID SULFOTRANSFERASE SULT2A1 AND RAT HYDROXYSTEROID SULFOTRANSFERASE STA

T. Idil Apak, and Michael W. Duffel

Division of Medicinal and Natural Products Chemistry, College of Pharmacy, The University of Iowa, Iowa City, Iowa

(Received June 8, 2004; accepted September 10, 2004)


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Tamoxifen (TAM) is a nonsteroidal antiestrogenic drug that is widely used for the treatment of estrogen receptor-dependent breast cancer. An increased risk of endometrial cancer in some patients treated with TAM has been linked to the metabolic formation of {alpha}-hydroxytamoxifen ({alpha}-OHTAM) and its subsequent sulfation. {alpha}-OHTAM has been found to be a substrate for rat and human hydroxysteroid sulfotransferases (STa and SULT2A1, respectively). Since stereochemistry plays an important role in the interactions of hydroxysteroid sulfotransferases with their substrates, we have now investigated the interactions of each of the stereoisomers of {alpha}-OHTAM with highly purified recombinant STa and SULT2A1. Methods for the preparation of the enantiomers of E- and Z-{alpha}-OHTAM were developed. When each of the four enantiomers was examined with rat STa, E-(+)-{alpha}-OHTAM was the only substrate for the enzyme, whereas E-(-)-{alpha}-OHTAM, Z-(+)-{alpha}-OHTAM, and Z-(-)-{alpha}-OHTAM were inhibitors of the sulfation of E-(+)-{alpha}-OHTAM catalyzed by STa. The dissociation constants for the {alpha}-OHTAM enantiomers indicated that they bound to STa with similar affinity, but only the E-(+)-enantiomer was a substrate. In contrast to the results obtained with rat hydroxysteroid sulfotransferase STa, all enantiomers of {alpha}-OHTAM were substrates for the human SULT2A1. Moreover, kcat/Km values with SULT2A1 were higher with the Z enantiomers than with the E enantiomers. As a result of the potential for interconversion of the E and Z geometric isomers upon metabolism, the sulfation of the Z isomers may be of greater concern in human tissues than has been previously assumed.

Tamoxifen (TAM) was one of the first selective estrogen modulators, and it is widely used for the adjuvant treatment of estrogen receptor-dependent breast cancer (Smith et al., 2000Go; Jordan, 2003aGo). Although it is a very successful chemotherapeutic agent, there is an increased risk of endometrial cancer in postmenopausal women treated with this drug (Assikis et al., 1996Go; O'Regan et al., 1998Go; Bernstein et al., 1999Go). Moreover, the use of TAM as a chemopreventive agent in patients at high risk for developing breast cancer also adds to the interest in understanding the mechanism(s) involved in this carcinogenic side effect (White, 1999Go; Smith et al., 2000Go). Numerous previous studies have implicated the activation of TAM to metabolites that covalently bind DNA as being critical components in the carcinogenic effects of this agent (Dasaradhi and Shibutani, 1997Go; Hardcastle et al., 1998Go; Fan and Bolton, 2001Go; Notley et al., 2002Go; Liu et al., 2003Go).

TAM is initially metabolized in reactions catalyzed by cytochrome P450 monooxygenases and flavin-containing monooxygenases (Mani et al., 1993Go; Kupfer and Dehal, 1996Go; Crewe et al., 2002Go). Several major metabolites of TAM are N-desmethylTAM, TAM-N-oxide, 4-hydroxyTAM, and {alpha}-hydroxytamoxifen ({alpha}-OHTAM). Some of these oxidized metabolites of TAM are further metabolized in reactions catalyzed by sulfotransferases and UDP-glucuronosyltransferases (Poon et al., 1993Go; Shibutani et al., 1998aGo; Boocock et al., 2000Go; Chen et al., 2002Go; Nishiyama et al., 2002Go).

Sulfotransferases catalyze the transfer of a sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to an acceptor molecule, thereby forming a sulfuric acid ester. Although sulfation is most often a component in metabolic pathways for the detoxication of xenobiotics, sulfation of the {alpha}-hydroxylated metabolites of TAM is considered to be an activation pathway due to DNA adduct formation following the reaction with the unstable sulfuric acid esters formed (Phillips et al., 1994Go; Potter et al., 1994Go; Randerath et al., 1994Go; Dasaradhi and Shibutani, 1997Go; Davis et al., 1998Go; Shibutani et al., 1998aGo,bGo; Surh, 1998Go).

Hydroxysteroid (alcohol) sulfotransferase STa, also systematically named either as ST2A2 (Nagata and Yamazoe, 2000Go) or (RAT)SULT2A3 (Blanchard et al., 2004Go), is a major sulfotransferase that catalyzes sulfation of xenobiotic alcohols in the liver and other tissues of the rat (Lyon and Jakoby, 1980Go; Ogura et al., 1990Go). It has previously been shown that {alpha}-OHTAM was a substrate for a homogeneous preparation of STa and that sulfation of E-{alpha}-OHTAM resulted in the formation of DNA adducts (Shibutani et al., 1998aGo). The human hydroxysteroid sulfotransferase SULT2A1 [also known as human dehydroepiandrosterone (DHEA) sulfotransferase, DHEA-ST, or ST2A3] has a 63% identity and 77% similarity to STa in its amino acid sequence, and it is the major enzyme catalyzing sulfation of DHEA in humans (Falany et al., 1995Go; Chang et al., 2001Go). Although cytosolic preparations of recombinant human SULT2A1 have been shown to catalyze sulfation of E-{alpha}-OHTAM (Shibutani et al., 1998bGo), there are apparently conflicting results reported on the mutagenicity of {alpha}-OHTAM in bacterial and mammalian cells engineered to express SULT2A1 and STa (Glatt et al., 1998Go).

Although E-{alpha}-OHTAM is the initial metabolite formed from the metabolic oxidation of TAM at the allylic position, an interconversion between E- and Z-{alpha}-OHTAM is possible as a result of sulfation and the subsequent reaction with water (Dasaradhi and Shibutani, 1997Go). Therefore, it is important to determine the potential for the formation of reactive sulfuric acid esters from both the E and Z isomers of {alpha}-OHTAM. Moreover, the chiral center at the {alpha} position on the ethyl side chain of {alpha}-OHTAM means that there are potentially two enantiomers for each of its two geometric isomers. R and S enantiomers of E-{alpha}-OHTAM have been resolved by Osborne et al. (2001Go), and their studies have shown that the R-(+)-isomer formed more adducts with DNA in hepatocytes than the S-(-)-isomer. However, the interactions of the enantiomers of both E- and Z-{alpha}-OHTAM with purified sulfotransferases (e.g., rat STa and human SULT2A1) have not been investigated. The present study explores the interactions of these four enantiomers of {alpha}-OHTAM with the purified hydroxysteroid sulfotransferases STa and SULT2A1.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and Instruments. Thin-layer chromatography (TLC) plates (250 µm, Silica Gel GF) were obtained from Analtech (Newark, DE). Flash chromatography was performed with silica gel (particle size, 32-63 µm) from Scientific Adsorbents Incorporated (Atlanta, GA). PAPS was obtained from Sigma-Aldrich (St. Louis, MO) and purified upon arrival using a published procedure (Sekura, 1981Go) to obtain 99% purity by HPLC. Subtilisin from Bacillus licheniformis and Candida antarctica B lipase was obtained from Altus Biologics Inc. (Cambridge, MA). HPLC experiments were performed on a Shimadzu LC-10AD instrument equipped with a Shimadzu UV spectrophotometer detector and an Econosphere C-18 (5 µm, 4.6 x 250 mm) reversed-phase column (Alltech Associates, Deerfield, IL). 1H NMR data were obtained with Bruker WM-360 and DRX-400 spectrometers (Bruker, Newark, DE). Determinations of optical activity were made with a Jasco model P-1020 polarimeter (Jasco, Tokyo, Japan). Fluorescence studies were performed with a PerkinElmer LS55 Luminescence Spectrometer (PerkinElmer Life and Analytical Sciences, Boston, MA). GC-MS was performed with a ThermoQuest Voyager 2000 series instrument (Thermo Finnigan, San Jose, CA) equipped with 15-m ZB-1 column. The ramp was held at 50°C for 1 min and then increased at 10°C/min and held at 250°C for 8 min.

Synthesis of E and Z-(±)-{alpha}-OHTAM. E- and Z-(±)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-buten-3-ol were synthesized by a published procedure (Foster et al., 1985Go), with t-butyllithium substituting for n-butyllithium. t-Butyllithium (3.7 mmol, 2.17 ml of a 1.7 M solution in pentane) was added to a stirred solution of E/Z-1-bromo-2-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenylene (0.781 g, 1.85 mmol) in dry tetrahydrofuran (10 ml) at -78°C under N2; this was followed after 5 min by acetaldehyde (0.61 ml, 10.9 mmol). The mixture was allowed to attain 0°C during 20 min and then quenched with H2O (2 ml) and partitioned between Et2O (20 ml) and H2O (20 ml). The Et2O layer was washed with H2O (15 ml), dried (Na2SO4), and concentrated. The crude material was chromatographed on a silica gel column (30 x 2 cm). The column was eluted first with 100 ml of hexane/Et2O/TEA (3:3:0.5) and then with 500 ml of hexane/Et2O/TEA (3:3:2) to give E- and Z-{alpha}-OHTAM. Z-{alpha}-OHTAM (yield = 26%), 1H NMR (360 MHz, CDCl3) {delta} (ppm): 1.24 [d, 3H, J = 6.4 Hz, -CH(OH)CH3], 2.40 [s, 6H, -N(CH3)2], 2.84 [t, 2H, J = 6.0 Hz, -CH2-N(CH3)2], 4.17 [t, 2H, J = 6.0 Hz, -O-CH2-], 4.88 [q, 1H, J = 6.6 Hz, CH(OH)CH3], 6.91 to 7.56 [m, 14H, 3 Ph]; and 13C NMR (90 MHz, CDCl3) {delta} (ppm): 22.53 [CH(OH)CH3], 46.08 [-N(CH3)2], 57.74 [-CH2-N(CH3)2], 67.78 [-O-CH2], 67.90 [-CH(OH)CH3], and 111 to 154 [aromatic and olefin carbons]. GC-MS: m/z 387 (M{bullet}+), 386 (M-H), 368 (M-H2O), 72 (C4H12N+), 58 (C3H8N+). E-{alpha}-OHTAM (yield = 35%), 1H NMR (360 MHz, CDCl3) {delta} (ppm): 1.19 [d, 3H, J = 6.0 Hz, -CH(OH)CH3], 2.31 [s, 6H, -N(CH3)2], 2.70 [t, 2H, J = 6.6 Hz, -CH2-N(CH3)2], 3.96 [t, 2H, J = 6.0 Hz, -O-CH2-], 4.80 [q, 1H, J = 6.2 Hz, CH(OH)CH3], 6.50 [d, 2H, J = 8.8 Hz, H-3,5 of C6H4O], 6.77 [d, 2H, J = 8.8 Hz, H-2,6 of C6H4O], 6.81 to 7.43 [m, 10H, 2Ph]; and 13C NMR (90 MHz, CDCl3) {delta} (ppm): 22.63 [-CH(OH)CH3], 46.30 [-N(CH3)2], 57.98 [-CH2-N(CH3)2], 67.75 [-O-CH2], 68.04 [-CH(OH)CH3], and 111 to 142 [aromatic and olefin carbons]. GC-MS: m/z 387 (M{bullet}+), 386 (M-H), 368 (M-H2O), 72 (C4H12N+), 58 (C3H8N+).

Synthesis of E- and Z-{alpha}-AcetoxyTAM. A catalytic amount of 4,4-dimethylaminopyridine was added to a stirred solution of either E-{alpha}-OHTAM or Z-{alpha}-OHTAM (0.10 g, 0.26 mmol) in 10 ml of dry triethylamine. To this solution, 2 ml of acetic anhydride was added, and the reaction was stirred for 1 h at room temperature. The reaction mixture was then treated with saturated NaHCO3 solution and extracted with methylene chloride (2 x 20 ml). The methylene chloride fractions were pooled, and the solvent was removed with a rotary evaporator to give either E- or Z-(±)-{alpha}-acetoxyTAM. E-(±)-{alpha}-AcetoxyTAM (yield = 95%), 1H NMR (360 MHz, CDCl3) {delta} (ppm): 1.48 [d, J = 6.6 Hz, -CH(OAc)CH3], 2.01 [s, 3H, -COCH3], 2.35 [s, 6H, N(CH3)2], 2.77 [t, 2H, J = 5.7 Hz, -CH2-N(CH3)2], 4.10 [t, 2H, J = 6.0 Hz, -O-CH2-], 6.19 [q, 1H, J = 6.6 Hz, CH(OAc)CH3], 6.78 [d, 2H, J = 8.6 Hz, H-3,5 of C6H4O], 7.01 [d, 2H, J = 8.2 Hz, H-2,6 of C6H4O], 7.04 to 7.39 (m, 10H, 2 Ph); and 13C NMR (90 MHz, CDCl3) {delta} (ppm): 20.91 [CH(OAc)CH3], 21.25 [-COCH3], 46.04 [-N(CH3)2], 58.17 [-CH2-N(CH3)2], 66.99 [-CH(OAc)CH3], 67.24 [-O-CH2-], 111 to 142 [aromatic and olefin carbons], and 170.19 [C = O]. GC-MS: m/z 429 (M{bullet}+), 370 (M-C2H3O2), 72 (C4H12N+), 58 (C3H8N+). Z-(±)-{alpha}-AcetoxyTAM (yield = 95%), 1H NMR (360 MHz, CDCl3) {delta} (ppm): 1.31 [d, 3H, J = 7.5 Hz, -CH(OAc)CH3], 1.92 [s, 3H, -COCH3], 2.40 [s, 6H, -N(CH3)2], 2.85 [t, 2H, J = 6.1 Hz, -CH2-N(CH3)2], 4.16 [t, 2H, J = 6.0 Hz, -O-CH2-], 5.79 [q, 1H, J = 6.6 Hz, CH(OAc)CH3], 6.84 to 7.52 [m, 14H, 3 Ph]; 13C NMR (90 MHz, CDCl3) {delta} (ppm): 19.74 [-CH(OAc)CH3], 21.29 [-COCH3], 46.22 [-N(CH3)2], 57.97 [-CH2-N(CH3)2], 67.85 [-CH(OAc)CH3], 71.42 [-O-CH2-], 112 to 154 [aromatic and olefinic carbons], and 170.09 [C = O]. GC-MS: m/z 429 (M{bullet}+), 370 (M-C2H3O2), 72 (C4H12N+), 58 (C3H8N+).

Synthesis of Z-(-)-{alpha}-OHTAM. Z-(±)-{alpha}-AcetoxyTAM (0.1 g) in 0.5 ml of acetone was added dropwise to a solution of 3 g of subtilisin from B. licheniformis in 60 ml of 0.1 M potassium phosphate buffer (pH 7). The reaction was stirred for up to 24 h and monitored by TLC (CH2Cl2/Et2O/TEA, 8:2:1) for the hydrolysis of the ester to the alcohol. At a point before 50% conversion from the ester to the alcohol had occurred, the reaction was stopped by extraction with CH2Cl2, and Z-(-)-{alpha}-OHTAM was obtained by silica gel flash chromatography, eluting first with hexane/Et2O/TEA (3:3:0.5) and then with hexane/Et2O/TEA (3:3:2). Yield = 23%. [{alpha}] = -74.5 c = 1, MeOH. 1H NMR (360 MHz, CDCl3) {delta} (ppm): 1.24 [d, 3H, J = 6.1 Hz, -CH(OH)CH3], 2.42 [s, 6H, -N(CH3)2], 2.86 [t, 2H, J = 6.7 Hz, -CH2-N(CH3)2], 4.19 [t, 2H, J = 6.1 Hz, -O-CH2-], 4.87 [q, 1H, J = 6.7 Hz, CH(OH)CH3], 6.84 to 7.52 [m, 14H, 3 Ph]; and 13C NMR (90 MHz, CDCl3) {delta} (ppm): 22.52 [-CH(OH)CH3], 45.97 [-N(CH3)2], 57.81 [-CH2-N(CH3)2], 65.16 [-O-CH2], 67.76 [-CH(OH)CH3], and 110 to 150 ppm [aromatic and olefin carbons]. GC-MS: m/z 387 (M{bullet}+), 386 (M-H), 368 (M-H2O), 72 (C4H12N+), 58 (C3H8N+).

Synthesis of Z-(+)-{alpha}-OHTAM. The unhydrolyzed ester [Z-(+)-{alpha}-acetoxyTAM] (3.3 mg, 0.0076 mmol) from the reaction to obtain Z-(-)-{alpha}-OHTAM was dissolved in 5 ml of MeOH/H2O (8:2) with a catalytic amount of K2CO3 and stirred for 16 h at room temperature. Following the reaction, methanol was removed with a rotary evaporator, and the residue was extracted with methylene chloride. The methylene chloride layers were pooled, and the solvent was removed with a rotary evaporator to give Z-(+)-{alpha}-OHTAM. Yield = 34%. [{alpha}] = +77.1 c = 0.14, MeOH. 1H NMR (360 MHz, CDCl3) {delta} (ppm): 1.24 [d, 3H, J = 6.4 Hz, -CH(OH)CH3], 2.43 [s, 6H, -N(CH3)2], 2.90 [t, 2H, J = 6.2 Hz, -CH2-N(CH3)2], 4.19 [t, 2H, J = 6.2 Hz, -O-CH2-], 4.90 [q, 1H, J = 6.4 Hz, -CH(OH)CH3], 6.84-7.51 [m, 14H, 3 Ph]; and 13C NMR (90 MHz, CDCl3) {delta} (ppm): 22.52 [-CH(OH)CH3], 45.98 [-N(CH3)2], 57.82 [-CH2-N(CH3)2], 67.86 [-O-CH2], 69.48[-CH(OH)CH3], and 110 to 150 ppm [aromatic and olefin carbons]. GC-MS: m/z 387 (M{bullet}+), 386 (M-H), 368 (M-H2O), 72 (C4H12N+), 58 (C3H8N+).

Synthesis of E-(-)-{alpha}-OHTAM. E-{alpha}-AcetoxyTAM (0.1 g) in 0.5 ml of acetone was added dropwise to a solution of 3 g of B. licheniformis subtilisin in 60 ml of 0.1 M potassium phosphate buffer (pH 7.0). The reaction mixture was stirred for up to 24 h and monitored by TLC (CH2Cl2/Et2O/TEA, 8:2:1) for the hydrolysis of the ester to the alcohol. The reaction was stopped by extraction with CH2Cl2 before 50% conversion of the ester to the alcohol had occurred, and E-(-)-{alpha}-OHTAM was obtained by silica gel flash chromatography using hexane/Et2O/TEA (3:3:0.5) followed by hexane/Et2O/TEA (3:3:2) to elute the product. Yield = 5.3%. [{alpha}] = -94.7 c = 1 (MeOH). 1H NMR (360 MHz, CDCl3) {delta} (ppm): 1.20 [d, 3H, J = 6.6 Hz, -CH(OH)CH3], 2.34 [s, 6H, -N(CH3)2], 2.75 [t, 2H, 6.7 Hz, -CH2-N(CH3)2], 3.99 [t, 2H, 6.7 Hz, -O-CH2-], 4.82 [q, 1H, J = 6.6 Hz, -CH(OH)CH3], 6.51 [d, 2H, J = 8.7 Hz, H-3,5 of C6H4O], 6.78 [d, 2H, J = 8.0 Hz, H-2,6 of C6H4O], 6.82 to 7.43 [m, 10H, 2Ph]; and 13C NMR (90 MHz, CDCl3) {delta} (ppm): 22.33 [-CH(OH)CH3], 45.95 [-N(CH3)2], 57.67 [-CH2-N(CH3)2], 67.45 [-O-CH2], 67.80 [-CH(OH)CH3], and 110 to 140 ppm [aromatic and olefin carbons]. GC-MS: m/z 387 (M{bullet}+), 386 (M-H), 368 (M-H2O), 72 (C4H12N+), 58 (C3H8N+).

Synthesis of E-(+)-{alpha}-OHTAM. E-(±)-{alpha}-AcetoxyTAM (0.1 g) in 0.5 ml of acetone was added dropwise to a solution of 3 g of C. antarctica B lipase in 60 ml of 0.1 M potassium phosphate buffer (pH 7.0). The reaction mixture was stirred for up to 24 h and monitored by TLC (CH2Cl2/Et2O/TEA, 8:2:1) for the hydrolysis of the ester to the alcohol. At a point before 50% of the ester had been converted to the alcohol, the reaction was stopped by extraction with CH2Cl2, and E-(+)-{alpha}-OHTAM was obtained by silica gel flash chromatography using hexane/Et2O/TEA (3:3:0.5) followed by hexane/Et2O/TEA (3:3:2). Yield = 4.6%. [{alpha}] = +110.7 c = 1 (MeOH). 1H NMR (360 MHz, CDCl3) {delta} (ppm): 1.21 [d, 3H, J = 6.6 Hz, -CH(OH)CH3], 2.19 [s, 6H, -N(CH3)2], 2.77 [t, 2H, J = 5.8 Hz, -CH2-N(CH3)2], 3.99 [t, 2H, J = 6.0 Hz, -O-CH2-], 4.82 [q, 1H, J = 6.3 Hz, -CH(OH)CH3], 6.52 [d, 2H, J = 8.6 Hz, H-3,5 of C6H4O], 6.79 [d, 2H, J = 8.9 Hz, H-2,6 of C6H4O], 6.82 to 7.43 [m, 10H, 2Ph]; and 13C NMR (90 MHz, CDCl3) {delta} (ppm): 22.32 [-CH(OH)CH3], 46.00 [-N(CH3)2], 58.32 [-CH2-N(CH3)2], 67.32 [-O-CH2], 68.04 [-CH(OH)CH3], and 110 to 140 ppm [aromatic and olefin carbons]. GC-MS: m/z 387 (M{bullet}+), 386 (M-H), 368 (M-H2O), 72 (C4H12N+), 58 (C3H8N+).

Determination of the Percentage of Enantiomeric Excess. Enantiomeric excess (ee%) values were determined by the formation of the {alpha}-camphanate ester of each enantiomer and evaluation of 1H NMR spectra of the esters. The {alpha}-OHTAM enantiomer (61 mg, 0.16 mmol), 1S-camphanyl chloride (341 mg, 1.6 mmol), dry dimethylformamide (6 ml), and pyridine (0.1 ml) were mixed and stirred for 20 h at room temperature under anhydrous conditions. The reaction was treated with ice/water and extracted with ethyl ether. The camphanyl ester was recovered in 56% yield by silica gel flash chromatography with a mobile phase of hexane/Et2O/TEA (3:3:4). 1H NMR spectra of {alpha}-camphanyl esters of racemic {alpha}-OHTAM and its enantiomers were used for the calculation of the ee% values with the help of the following formula: ee% = [(E1 - E2)/(E1 + E2)] x 100%, where E1 = fraction of enantiomer 1 and E2 = fraction of enantiomer 2. The camphanyl ester of the racemic E-(±)-{alpha}-OHTAM showed two sets of quartets with equal intensity at 5.88 and 5.92 ppm due to the coupling of the methine proton to the adjacent methyl group. On the other hand, the camphanyl esters of E-(-)-{alpha}-OHTAM and E-(+)-{alpha}-OHTAM each displayed only one set of signals at this range (at 5.88 and 5.92 ppm, respectively). For racemic Z-(±)-{alpha}-OHTAM, the camphanyl esters showed two sets of signals at 5.98 and 6.03 ppm. The camphanyl esters of Z-(+)-{alpha}-OHTAM and Z-(-)-OHTAM each displayed only one set of signals (at 5.98 and 6.03 ppm, respectively). The ee% values were calculated from the integration of these resonances in the 1H NMR spectra.

STa and SULT2A1 Preparation. Expression and purification of recombinant rat STa and human SULT2A1 were performed according to published procedures (Sheng and Duffel, 2001Go, 2003Go). After expression in Escherichia coli BL21 (DE3) cells, the sulfotransferase was purified to apparent homogeneity by adenosine-3',5'-diphosphate (PAP)-agarose affinity chromatography. The excess PAP in the enzyme solution was removed by ultrafiltration (PM-10 membrane; Millipore Corporation, Bedford, MA). The protein concentrations were determined by the modified Lowry procedure, with bovine serum albumin as the standard (Bensadoun and Weinstein, 1976Go). During purification, the enzyme activity was determined using a methylene blue paired ion extraction method with DHEA as the substrate (Sheng et al., 2001Go).

Assay of Racemate and Enantiomers of {alpha}-OHTAM with STa and SULT2A1. Racemic {alpha}-OHTAM and its four stereoisomers were examined for their interactions with rat STa and human SULT2A1 using an established HPLC procedure (Duffel et al., 1989Go; Sheng et al., 2001Go). A typical reaction mixture contained a final concentration of 0.20 mM PAPS, 8.0 mM 2-mercaptoethanol, and 0.25 M potassium phosphate buffer. Reactions (final volume, 30 µl) were started by the addition of 0.8 to 1 µg of enzyme, incubated at 37°C for 15 min, and stopped by the addition of 30 µl of methanol. Substrate-dependent formation of PAP from PAPS in the reaction was determined by HPLC as described previously (Duffel et al., 1989Go). HPLC analysis of each reaction mixture was carried out in duplicate. Calculation of apparent Km and Vmax values was carried out by nonlinear least-squares fit to the equation v = Vmax x [S]/(Km + [S]).

Kinetics of Inhibition. Four concentrations of E-(+)-{alpha}-OHTAM (60, 120, 200, and 400 µM) were used as the substrate in the presence of 0.20 mM PAPS. E-(-)-, Z-(-)-, and Z-(+)-{alpha}-OHTAM were used as potential inhibitors of E-(+)-{alpha}-OHTAM at concentrations of 80, 200, and 400 µM. Substrates and inhibitors were mixed to give the final concentrations above. Data were fitted to rate equations for competitive, noncompetitive, or uncompetitive inhibition using the nonlinear least-squares algorithm in the Enzyme Kinetics Module 1.1 of Sigma Plot 8.0, and the model with the highest value for the coefficient of determination, r2, was selected.

Determination of Dissociation Constants. Dissociation constants (Kd) for each stereoisomer were calculated by observing the decrease in intrinsic fluorescence of the enzyme at 347 nm upon excitation at 290 nm. Reaction cuvettes were kept at 25°C, and each cuvette contained 9.0 mM 2-mercaptoethanol, 0.25 M potassium phosphate buffer (pH 7.0), and 50 nM STa. All solutions were filtered with a Millex-GS 0.22 µM filter unit (Millipore Corporation) before analysis. Each ligand was added in 0.5-µl aliquots, and the solution was stirred with a Teflon rod after each addition. Fluorimeter shutters were closed unless spectra were being recorded to minimize UV exposure time. The decrease in intrinsic fluorescence of the protein was measured at 347 nm after each addition of the ligand. The data were corrected for the small dilution occurring when aliquots of the ligand were added.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Synthesis of Stereoisomers of {alpha}-OHTAM. Although a method for the combination of chemical synthesis and HPLC separation to obtain enantiomers of E-{alpha}-OHTAM has been reported earlier (Osborne et al., 2001Go), the synthesis of all four enantiomers of E- and Z-{alpha}-OHTAM has not been previously described. Our initial attempts to obtain these stereoisomers of E- and Z-{alpha}-OHTAM by the oxidation of these {alpha}-OHTAMs to a prochiral ketone followed by reduction with commercially available enantioselective reducing agents were not successful (data not shown). Because of this, we developed a method to obtain the stereoisomers of {alpha}-OHTAM through a combination of chemical and biocatalytic approaches.

We first prepared both E- and Z-{alpha}-OHTAM in the racemic forms by using a previously published procedure (Foster et al., 1985Go), wherein we substituted t-butyllithium for n-butyllithium in the reaction of 1-bromo-2-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenylethylene with acetaldehyde. Yields for E- and Z-{alpha}-OHTAM with this reagent were 35 and 26%, respectively, and these compared favorably to the published yields with n-butyllithium of 33 and 22%, respectively (Foster et al., 1985Go). However, since our observed yields of the E and Z isomers with n-butyllithium were 18 and 28%, respectively, we used t-butyllithium for all subsequent preparations of {alpha}-OHTAM.

To obtain the stereoisomers of {alpha}-OHTAM, we first acetylated E- and Z-{alpha}-OHTAM to produce E- and Z-{alpha}-acetoxyTAM in high yield (Fig. 1). After initial screening with a total of 30 esterases and lipases, we identified two enzymes that were suitable for stereospecific hydrolysis of the E- and Z-{alpha}-acetoxyTAM: C. antarctica B lipase and subtilisin from B. licheniformis. These two hydrolytic enzymes were used for obtaining E-(-)-, E-(+)-, and Z-(-)-enantiomers as described under Materials and Methods. With this method, E-(-)- and Z-(-)-{alpha}-OHTAM could be obtained by the hydrolysis of the respective E-{alpha}-acetoxyTAM and Z-{alpha}-acetoxyTAM catalyzed by subtilisin from B. licheniformis (Fig. 1). We were able to obtain E-(+)-{alpha}-OHTAM with C. antarctica B lipase by the hydrolysis of E-{alpha}-acetoxyTAM (Fig. 1); however, Z-(+)-{alpha}-OHTAM could not be obtained from Z-{alpha}-acetoxyTAM with the same enzyme. Therefore, we obtained the Z-(+)-isomer of {alpha}-OHTAM by the chemical hydrolysis of the unreacted ester [Z-(+)-{alpha}-acetoxyTAM] remaining in the reaction mixture after the completion of the subtilisin-catalyzed hydrolysis of Z-{alpha}-acetoxyTAM and subsequent removal of the Z-(-)-{alpha}-OHTAM. (Fig. 1). The ee% value of each stereoisomer was determined after forming the camphanyl derivative, and the results are shown in Table 1. These four enantiomers were then examined for their interactions with rat STa and human SULT2A1.



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  		FIG. 1. Chiral resolution of {alpha}-OHTAM stereoisomers. Reagents or enzyme required for each step are as follows: a) TEA, 4,4-dimethylaminopyridine, and acetic anhydride; b) subtilisin from B. licheniformis; c) lipase B from C. antarctica; and d) methanol/H2O (8:2), K2CO3.

 

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  		TABLE 1 Summary of the specific rotation and enantiomeric excess of {alpha}-OHTAM stereoisomers

Values of ee were determined by the formation of the {alpha}-camphanate ester of each enantiomer and evaluation of 1H NMR spectra of the esters.

 

Stereoisomers of {alpha}-OHTAM as Substrates for Rat STa and Human SULT2A1. Both rat STa and human SULT2A1 were previously shown to be either stereoselective or stereospecific in catalyzing the sulfation of chiral secondary alcohols (Banoglu and Duffel, 1997Go, 1999Go; Sheng and Duffel, 2003Go). Based on these findings, we examined purified rat STa and human SULT2A1 for their ability to catalyze sulfation of each of the E- and Z-{alpha}-OHTAM stereoisomers The maximal velocity (Vmax), Michaelis constant (Km), and catalytic efficiency (kcat/Km) obtained from these studies with rat STa and human SULT2A1 are shown in Tables 2 and 3, respectively. As seen in Table 2, E-(+)-{alpha}-OHTAM was a better substrate for rat STa than racemic E-{alpha}-OHTAM, with an approximately 2-fold higher kcat/Km. None of the stereoisomers of Z-{alpha}-OHTAM served as substrates for the rat STa. When the stereoisomers of {alpha}-OHTAM were examined with human SULT2A1, all stereoisomers were substrates for the enzyme. The kinetic parameters that were obtained from these studies with human SULT2A1 are shown in Table 3.


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  		TABLE 2 Sulfation of {alpha}-OHTAM stereoisomers catalyzed by rat STa

Calculation of kcat values was based on 33,120 as the subunit molecular mass of STa.

 

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  		TABLE 3 Sulfation of the stereoisomers of {alpha}-OHTAM catalyzed by human SULT2A1

Calculation of kcat values was based on 33,648 as the subunit molecular mass of SULT2A1.

 

Inhibition Studies. Since E-(-)-, Z-(-)-, and Z-(+)-{alpha}-OHTAM were not substrates for reactions catalyzed by rat STa, these stereoisomers were examined as potential inhibitors of the sulfation of E-(+)-{alpha}-OHTAM catalyzed by the enzyme. The nonsubstrate stereoisomers were inhibitors of the STa-catalyzed sulfation of E-(+)-{alpha}-OHTAM, and Ki values were 730, 650, and 100 µM for E-(-)-{alpha}-OHTAM, Z-(-)-{alpha}-OHTAM, and Z-(+)-{alpha}-OHTAM, respectively. E-(-) and Z-(-)-{alpha}-OHTAM were noncompetitive inhibitors (Fig. 2, A and B), and Z-(+)-{alpha}-OHTAM was a competitive inhibitor (Fig. 2C) of the sulfation of E-(+)-{alpha}-OHTAM catalyzed by STa. Although we were able to determine the type of inhibition and Ki values for the nonsubstrate enantiomers, we wanted to understand these interactions of the enzyme with substrates and inhibitors more fully. This was accomplished by determining the dissociation constants for the binding of each stereoisomer to STa in the absence of PAPS.



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  		FIG. 2. Inhibition of the sulfation of E-(+)-{alpha}-OHTAM catalyzed by rat STa. For each inhibition study, the following concentrations of inhibitor were used: , no inhibitor; {circ}, 80 µM; {blacktriangledown}, 200 µM; and {triangledown}, 400 µM. A, noncompetitive inhibition by E-(-)-{alpha}-OHTAM with a Ki determined to be 730 µM. B, noncompetitive inhibition by Z-(-)-{alpha}-OHTAM with a Ki determined to be 650 µM. C, competitive inhibition by Z-(+)-{alpha}-OHTAM with a Ki determined be 100 µM.

 

Binding of {alpha}-OHTAM Stereoisomers to STa. Dissociation constants (Kd) of the stereoisomers of E- and Z-{alpha}-OHTAM were determined by using the decrease in the intrinsic fluorescence of rat STa upon binding of the molecules. The difference in the fluorescence emission at 347 nm ({Delta}F) was calculated after the addition of each stereoisomer and plotted as a function of the concentration of the {alpha}-OHTAM stereoisomer (Fig. 3). The dissociation constant (Kd) was calculated with the following equation, where [S] is the concentration of the stereoisomer of {alpha}-OHTAM: {Delta}F = {Delta}Fmax x [S]/(Kd + [S]).



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  		FIG. 3. Determination of dissociation constants for the stereoisomers of {alpha}-OHTAM with rat STa. The absolute value of the decrease in intrinsic fluorescence of the enzyme at 347 nm (excitation at 290 nm) is represented by {Delta}F. Dissociation constants were as follows: 50 ± 6 µM for E-(+)-{alpha}-OHTAM (A); 47 ± 4 µM for E-(-)-{alpha}-OHTAM (B); 67 ± 4 µM for Z-(+)-{alpha}-OHTAM (C); and 64 ± 8 µM for Z-(-)-{alpha}-OHTAM (D).

 

Our results showed that the Kd values for the binding of the stereoisomers of {alpha}-OHTAM to STa were between 47 and 67 µM (Fig. 3). The Kd of Z-(+)-{alpha}-OHTAM is close to its Ki value, and this correlates well with a competitive inhibition pattern, which indicates a direct interaction of the inhibitor solely with the sulfuryl acceptor-binding site on the free enzyme. On the other hand, the Kd values of E-(-)-{alpha}-OHTAM and Z-(-)-{alpha}-OHTAM are much lower than their respective Ki values. This is consistent with the noncompetitive inhibition pattern of these stereoisomers, wherein the inhibitor binds to both the free enzyme (as measured in the intrinsic fluorescence studies) and an enzyme-substrate complex.


   Discussion
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 Abstract
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References
 
The triphenylethylenic antiestrogen TAM is widely used for the treatment of breast cancer and is currently being used as a chemopreventive agent for women at high risk for developing breast cancer (Fisher et al., 1998Go; Jordan, 2003aGo,bGo,cGo). However, TAM is a potent carcinogen in rat liver (Williams and Iatropoulos, 1993Go), and an increased incidence of endometrial cancer has been detected in certain populations of women receiving TAM (Fisher et al., 1994Go). Attempts to detect TAM-DNA adducts in the endometria of women treated with TAM have been reported by several research groups (Carmichael et al., 1996Go; Shibutani et al., 1999Go). Shibutani and coworkers successfully detected these adducts in the endometrium through the use of a highly sensitive method of 32P postlabeling coupled with HPLC separations (Shibutani et al., 1999Go; Terashima et al., 1999Go), and they identified these metabolites as both trans and cis epimers of {alpha}-(N2-deoxyguanosinyl)-TAM. It has been suggested that these trans- and cis-DNA adducts can lead to the formation of the mutagenic effects in humans.

Earlier findings demonstrated that benzylic and allylic alcohols are activated upon sulfation by rat and human hydroxysteroid sulfotransferases (Miller et al., 1990Go; Ogura et al., 1990Go; Glatt et al., 1995Go). Because of the electron-withdrawing properties of the , the C-O bond can undergo heterolytic cleavage and, in the case of {alpha}-OHTAM, the resulting carbocation has been found to be sufficiently stable for DNA adduct formation (Kuramochi, 1996Go). As seen in Fig. 4, the carbocation is stabilized by resonance, such that either cis- or trans-DNA adducts may be obtained (Shibutani et al., 1998aGo). Although the two benzylic carbocation structures (resonance structures B and C in Fig. 4) would be highly stabilized, direct attack at the benzylic carbon (carbon-1) by nucleophilic sites on DNA would be sterically hindered. Nucleophilic addition could, however, occur at carbon-3 (i.e., either at the vinylic carbon of resonance structures B or C or at the localized carbocation in structures A or D of Fig. 4) to yield either cis or trans adducts. Alternatively, the reaction of the carbocation with water can lead to the formation of either E- or Z-{alpha}-OHTAM, thus providing both isomers in vivo.



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  		FIG. 4. Selected resonance structures involving carbocations that are formed following sulfation of E-{alpha}-OHTAM. Rotation of the bond between carbon-1 and carbon-2 in structures B and C gives rise to the possibility of either cis or trans products.

 

Since the stereochemistry of many allylic and benzylic alcohols is an important factor in their interactions with sulfotransferases, this prompted us to investigate the interactions of rat and human hydroxysteroid sulfotransferases with each of the individual stereoisomers of {alpha}-OHTAM. In our initial efforts to obtain {alpha}-OHTAM stereoisomers, we used {alpha}-oxidation to obtain a prochiral ketone and attempted chiral reduction with common asymmetric reducing reagents. However, the reduction step proved to be unsuccessful, potentially due to either steric effects or the tertiary amine of the dimethylaminoethoxy portion of the molecule. Thus, we developed a procedure whereby each of the enantiomers of E- and Z-{alpha}-OHTAM was obtained by the combination of chemical synthesis and biocatalytic steps (Fig. 1 and Table 1).

Previous studies have suggested that rat STa is stereoselective in its interactions with a series of chiral benzylic alcohols (Banoglu and Duffel, 1997Go). In the case of rat STa, it was previously shown that the enzyme could distinguish between the two geometric isomers of E- and Z-{alpha}-OHTAM (Shibutani et al., 1998aGo). Our present results on the individual enantiomers of E- and Z-{alpha}-OHTAM are consistent with those previous studies on STa; that is, racemic E-{alpha}-OHTAM was a good substrate for rat STa. However, E-(+)-{alpha}-OHTAM was a better substrate (Table 2) and had a higher kcat/Km value than the racemate. These results agreed with earlier studies indicating that E-(+)-{alpha}-OHTAM formed more adducts in rat hepatocytes than E-(-)-{alpha}-OHTAM (Osborne et al., 2001Go). Interestingly, all other stereoisomers of {alpha}-OHTAM were not substrates for rat STa under the same conditions. Moreover, further investigations revealed that the nonsubstrate enantiomers were noncompetitive [E-(-)- and Z-(-)-{alpha}-OHTAM] and competitive [Z-(+)-{alpha}-OHTAM] inhibitors of the STa-catalyzed sulfation of E-(+)-{alpha}-OHTAM (Fig. 2).

In addition to the kinetic studies, binding studies were performed to determine the dissociation constants (Kd) of {alpha}-OHTAM stereoisomers with STa. These experiments were done by taking advantage of the intrinsic fluorescence of STa. The absolute value of the change in the fluorescence upon binding of each ligand was determined as a function of the concentration of added {alpha}-OHTAM stereoisomer (Fig. 3). Values for Kd obtained from these binding studies were between 47 and 67 µM for all {alpha}-OHTAM stereoisomers. Thus, in the case of E-(-)-{alpha}-OHTAM and Z-(-)-{alpha}-OHTAM, the Kd values were about 10-fold lower then their Ki values (Figs. 2 and 3). These results suggested the possibility of lower-affinity interactions with an enzyme-substrate complex (e.g., an enzyme-PAPS complex) involved in the noncompetitive inhibition observed with these two enantiomers. On the other hand, the Kd for Z-(+)-{alpha}-OHTAM was similar to its Ki value, and this was consistent with a direct competition for binding of this stereoisomer to the free enzyme (competitive inhibition). Further kinetic studies will be necessary to fully elucidate the differences in the interactions of the inhibitory stereoisomers with rat STa.

Although previous studies have shown some similarity between rat STa and human SULT2A1 in the stereospecificity of their interactions with chiral 1-naphthyl-1-ethanols (Sheng and Duffel, 2003Go), it was still important to investigate the stereospecificity of interactions with SULT2A1 for the more structurally complex {alpha}-OHTAM stereoisomers. The results of these studies showed that all stereoisomers of {alpha}-OHTAM were substrates for human SULT2A1, and racemic Z-{alpha}-OHTAM was the substrate with the highest catalytic efficiency, as indicated by the value of kcat/Km (Table 3). Thus, although the formation of inhibitory stereoisomers of {alpha}-OHTAM may limit the overall production of the reactive {alpha}-OHTAM sulfate in the case of rat STa, such a limitation does not occur for the human SULT2A1. Although the overall catalytic efficiencies of the human SULT2A1 with stereoisomers of E-{alpha}-OHTAM are somewhat lower than the catalytic efficiency of the rat STa with E-(+)-{alpha}-OHTAM, this may be compensated by both the higher catalytic efficiency with the Z isomers of {alpha}-OHTAM and the lack of inhibition of SULT2A1 by any of the stereoisomers of {alpha}-OHTAM.

The molecular basis for the observed differences between human SULT1A1 and rat STa in the stereospecificity of interactions with {alpha}-OHTAM remains to be determined. The human SULT2A1 and rat STa have about 63% identity and 77% similarity in amino acid sequence. Nonetheless, our preliminary homology modeling of rat STa and comparison with the crystal structure of human SULT2A1 indicates that several amino acids lining the sulfuryl acceptor region of the active site are different. Since the overall three-dimensional structures of the peptide bond backbones of sulfotransferases have a high degree of homology (Negishi et al., 2001Go; Yoshinari et al., 2001Go), such differences in specific amino acid side chains likely play an important role in the stereospecificity of rat STa with these TAM stereoisomers. Further studies on the functional implications of individual amino acid differences between the two enzymes will be required to discern the structural basis for differences in the stereospecificity of the two enzymes in their interactions with {alpha}-OHTAM.


   Footnotes
 
This research was supported by research Grant R01 CA38683 from the National Cancer Institute, National Institutes of Health.

doi:10.1124/dmd.104.000919.

ABBREVIATIONS: TAM, tamoxifen;{alpha}-OHTAM, {alpha}-hydroxytamoxifen; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; DHEA, dehydroepiandrosterone; TLC, thin-layer chromatography; HPLC, high-performance liquid chromatography; GC-MS, gas chromatography-mass spectometry; Et2O, diethyl ether; TEA, triethylamine; ee, enantiomeric excess; PAP, adenosine 3',5'-diphosphate.

Address correspondence to: Dr. Michael W. Duffel, Division of Medicinal and Natural Products Chemistry, College of Pharmacy, The University of Iowa, Iowa City, IA 52242. E-mail: michael-duffel{at}uiowa.edu


   References
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 Materials and Methods
 Results
 Discussion
 References
 


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S. Y. Kim, Y. R. S. Laxmi, N. Suzuki, K. Ogura, T. Watabe, M. W. Duffel, and S. Shibutani
FORMATION OF TAMOXIFEN-DNA ADDUCTS VIA O-SULFONATION, NOT O-ACETYLATION, OF {alpha}-HYDROXYTAMOXIFEN IN RAT AND HUMAN LIVERS
Drug Metab. Dispos., November 1, 2005; 33(11): 1673 - 1678.
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D. H. Phillips, A. Hewer, M. R. Osborne, K. J. Cole, C. Churchill, and V. M. Arlt
Organ specificity of DNA adduct formation by tamoxifen and {alpha}-hydroxytamoxifen in the