Sulfation of iodothyronines by recombinant human liver steroid sulfotransferases.

Sulfation is an important pathway in the metabolism of thyroid hormones. Sulfated iodothyronines are elevated in nonthyroidal illnesses and in the normal human fetal circulation. We assayed and characterized COS-1 cell expressed recombinant human liver dehydroepiandrosterone sulfotransferase (DHEA ST or SULT2A1) and estrogen sulfotransferase (EST or SULT1E1) activities for the first time with triiodothyronine (T(3)) as the substrate. Several biochemical properties that included apparent K(m) values, thermal stabilities, and responses to the inhibitors 2, 6-dichloro-4-nitrophenol and NaCl were tested. SULT2A1, a member of the hydroxysteroid sulfotransferase family, used 3,3'-T(2) more readily than T(3) and 3,5-T(2) as substrates, but had the lowest apparent K(m) value for T(3) of any reported human SULT. SULT1E1, a member of the phenol sulfotransferase family, used 3,3'-T(2) and rT(3) more readily than T(3), and also displayed the greatest specificity for T(4) among human SULTs. SULT2A1 may contribute more to iodothyronine sulfation than previously suspected. Potential roles of both steroid sulfotransferases in the enhanced sulfation of nonthyroidal illnesses and fetal development invite further investigation.     

Oral contraceptives as substrates and inhibitors for human cytosolic SULTs

Cytosolic sulfotransferases (SULTs) in mammals are involved in the biotransformation and homeostasis of various endogenous and xenobiotic compounds. The current study aimed to examine the sulfation of contraceptive compounds by various human cytosolic SULTs and to investigate the inhibitory effects and mode of action of these compounds on the sulfation of 17beta-estradiol, a major endogenous estrogen. A systematic study using all eleven known human cytosolic SULTs revealed the differential substrate specificity of these enzymes for the eight representative contraceptive compounds and two endogenous estrogens (estrone and 17beta-estradiol) tested as substrates. Activity data showed that SULT1A1 displayed the strongest activity toward 17alpha-ethynylestradiol. Kinetic studies revealed that the V (max) value of the sulfation of 17alpha-ethynylestradiol by SULT1A1 was 1.64 times that of the sulfation of 17beta-estradiol, while the K (m) values were almost equal for the two compounds. The inhibitory effects of three contraceptive compounds on the sulfation of 17beta-estradiol by SULT1A1 were examined. IC(50) values determined were 0.193, 1.84, and 2.98 mM, respectively, for 19-norethindrone acetate, ethynodiol diacetate and mifepristone. Kinetic analyses indicated that the mechanism underlying the inhibition by these contraceptives is of a mixed noncompetitive type. Metabolic labeling experiments confirmed the sulfation of contraceptive compounds and the release of their sulfated derivatives by HepG2 human hepatoma cells. Collectively, the results obtained suggest a role of sulfation in the metabolism of contraceptive compounds in vivo. Moreover, in view of their inhibitory effects on the sulfation of 17beta-estradiol, these compounds may potentially act to disrupt the homeostasis of endogenous estrogens.     

Installation of O-glycan sulfation capacities in human HEK293 cells for display of sulfated mucins

The human genome contains at least 35 genes that encode Golgi sulfotransferases that function in the secretory pathway, where they are involved in decorating glycosaminoglycans, glycolipids, and glycoproteins with sulfate groups. Although a number of important interactions by proteins such as selectins, galectins, and sialic acid–binding immunoglobulin-like lectins are thought to mainly rely on sulfated O-glycans, our insight into the sulfotransferases that modify these glycoproteins, and in particular GalNAc-type O-glycoproteins, is limited. Moreover, sulfated mucins appear to accumulate in respiratory diseases, arthritis, and cancer. To explore further the genetic and biosynthetic regulation of sulfated O-glycans, here we expanded a cell-based glycan array in the human embryonic kidney 293 (HEK293) cell line with sulfation capacities. We stably engineered O-glycan sulfation capacities in HEK293 cells by site-directed knockin of sulfotransferase genes in combination with knockout of genes to eliminate endogenous O-glycan branching (core2 synthase gene GCNT1) and/or sialylation capacities in order to provide simplified substrates (core1 Galβ1–3GalNAcα1–O-Ser/Thr) for the introduced sulfotransferases. Expression of the galactose 3-O-sulfotransferase 2 in HEK293 cells resulted in sulfation of core1 and core2 O-glycans, whereas expression of galactose 3-O-sulfotransferase 4 resulted in sulfation of core1 only. We used the engineered cell library to dissect the binding specificity of galectin-4 and confirmed binding to the 3-O-sulfo-core1 O-glycan. This is a first step toward expanding the emerging cell-based glycan arrays with the important sulfation modification for display and production of glycoconjugates with sulfated O-glycans.     

Sulfation of "estrogenic" alkylphenols and 17beta-estradiol by human platelet phenol sulfotransferases

We have investigated the ability of alkylphenols to act as substrates and/or inhibitors of phenol sulfotransferase enzymes in human platelet cytosolic fractions. Our results indicate: (i) straight chain alkylphenols do not interact with the monoamine-sulfating phenol sulfotransferase (SULT1A3); (ii) short chain 4-n-alkylphenols (C < 8) are substrates for the phenol-sulfating enzymes (SULT1A1/2), which exhibit two activity maxima against substrates with alkyl chain lengths of C1-2 and C4-5; (iii) long chain 4-n-substituted alkylphenols (C >/= 8) are poor substrates and act as inhibitors of SULT1A1/2; (iv) human platelets contain two activities, of low and high affinity, capable of sulfating 17beta-estradiol, and 4-n-nonylphenol is a partial mixed inhibitor of the low affinity form of this activity. We conclude that by acting either as substrates or inhibitors of SULT1A1/2, alkylphenols may influence the sulfation, and hence the excretion, of estrogens and other phenol sulfotransferase substrates in humans.     

Differential roles of human monoamine (M)-form and simple phenol (P)-form phenol sulfotransferases in drug metabolism

Cytosolic sulfotransferases (STs) are traditionally known as Phase II drug-metabolizing or detoxifying enzymes that facilitate the removal of drugs and other xenobiotic compounds. In this study, we carried out a systematic investigation on the sulfation of drug compounds by two major human phenol STs (PSTs), the monoamine (M)-form and simple phenol (P)-form PSTs. Activity data obtained showed the differential substrate specificity of the two enzymes for the thirteen drug compounds tested. Kinetic studies revealed that the M-form PST displayed stereoselectivity for the chiral drug, isoproterenol. The effects of divalent metal cations on the activity of the M-form and P-form PSTs toward representative drug compounds were quantitatively evaluated. Results obtained indicated that the drug-sulfating activities of the two human PSTs were partially or completely inhibited or stimulated by the ten divalent metal cations tested at a 5 mM concentration. The two enzymes appeared to be less sensitive to the effects of physiologically more abundant metal cations such as Mg(2+) and Ca(2+), but more sensitive to the detrimental effects of other metal cations that may enter the body as environmental contaminants.     

Reaction product affinity regulates activation of human sulfotransferase 1A1 PAP sulfation

Cytosolic sulfotransferase (SULT)-catalyzed sulfation regulates the activity of bio-signaling molecules and aids in metabolizing hydroxyl-containing xenobiotics. The sulfuryl donor for the SULT reaction is adenosine 3′-phosphate 5′-phosphosulfate (PAPS), while products are adenosine 3′,5′-diphosphate (PAP) and a sulfated alcohol. Human phenol sulfotransferase (SULT1A1) is one of the major detoxifying enzymes for phenolic xenobiotics. The mechanism of SULT1A1-catalyzed sulfation of PAP by pNPS was investigated. PAP was sulfated by para-nitrophenyl sulfate (pNPS) in a concentration-dependent manner. 2-Naphthol inhibited sulfation of PAP, competing with pNPS, while phenol activated the sulfation reaction. At saturating PAP, a ping pong kinetic mechanism is observed with pNPS and phenol as substrates, consistent with phenol intercepting the E–PAPS complex prior to dissociation of PAPS. At high concentrations, phenol competes with pNPS, consistent with formation of the E–PAP–phenol dead-end complex. Data are consistent with the previously reported mechanism for sulfation of 2-naphthol by PAPS, and its activation by pNPS [14]. Overall, data are consistent with release of PAP from E–PAP and PAPS from E–PAPS contributing to rate-limitation in both reaction directions.     

Sulfation of flavonoids and other phenolic dietary compounds by the human cytosolic sulfotransferases

The protective effects of diet, especially soya products, tea, and many fruits, against a variety of human cancers, as suggested by epidemiological studies, has focused attention on flavonoids, isoflavonoids, and other phenolic dietary compounds as chemoprotectants. Among the mechanisms suggested for their chemoprotective action, their ability to inhibit the bioactivation of carcinogens by the human cytosolic sulfotransferases (STs) and the direct effects of their sulfoconjugates are being increasingly studied. We report here a systematic study on the sulfation of representative flavonoids, isoflavonoids, anti-oxidants, and other phenolic dietary compounds by all ten known human cytosolic STs. All ten recombinant human cytosolic STs were prepared in a pure form and tested for their sulfating activities with a variety of these compounds. P-form (SULT1A1) phenol ST (PST) showed high sulfating activity with most of these compounds. M-form (SULT1A3) PST showed high activity with the flavonoids but not with the isoflavonoids. SULT1C ST #2 showed high activity with the isoflavonoids and also sulfated most of the other compounds. Possible relevance of these results to the chemoprotective effects of these dietary compounds is discussed.     

Generation and release of nitrotyrosine O-sulfate by HepG2 human hepatoma cells upon SIN-1 stimulation: identification of SULT1A3 as the enzyme responsible

In addition to serving as a biomarker of oxidative/nitrative stress, elevated levels of nitrotyrosine have been shown to cause DNA damage or trigger apoptosis. Whether the body is equipped with mechanisms for protecting against the potentially harmful nitrotyrosine remains unknown. The present study was designed to investigate the possibility that sulfation serves as a pathway for the metabolism/regulation of nitrotyrosine. Using metabolic labelling, nitrotyrosine O-[35S]sulfate was found to be produced and released into the medium of HepG2 human hepatoma cells labelled with [35S]sulfate in the presence of nitrotyrosine. To identify the enzyme(s) responsible for nitrotyrosine sulfation, a systematic study of all eleven known human cytosolic SULTs (sulfotransferases) was performed. Of the 11 enzymes tested, only SULT1A3 displayed sulfating activity toward nitrotyrosine. The pH-dependence and kinetic constants of SULT1A3 with nitrotyrosine or dopamine as substrate were determined. To examine whether the sulfation of nitrotyrosine occurs in the context of cellular physiology, HepG2 cells labelled with [35S]sulfate were treated with SIN-1 (morpholinosydnonimine), a peroxynitrite generator. Increments of nitrotyrosine O-[35S]sulfate were detected in the medium of HepG2 cells treated with higher concentrations of SIN-1. To gain insight into the physiological relevance of nitrotyrosine sulfation, a time-course study was performed using [3H]tyrosine-labelled HepG2 cells treated with SIN-1. The findings confirm that the bulk of free [3H]nitrotyrosine inside the cells was present in the unconjugated form. The proportion of sulfated [3H]nitrotyrosine increased dramatically in the medium over time, implying that sulfation may play a significant role in the metabolism of free nitrotyrosine.     

Continuous fluorometric assay of phenol sulfotransferase.

Phenol sulfotransferases (EC 2.8.2.1) catalyze the sulfation of the acceptor hydroxyl group using 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the donor substrate. Previous assays of these enzymes, which exhibit varied acceptor substrate specificities, have required termination of the catalysis followed by isolation and quantitation of formed sulfate ester. In this report, the sulfation of the fluorescent compound, resorufin, is investigated. Reaction of PAPS with resorufin, catalyzed by bovine lung phenol sulfotransferase, bleaches the emission of this acceptor at the pH of the reaction (pH 6.4 optimum). It is thereby possible to continuously record the sulfation reaction. Analysis of single progress curves by integrated replot can be used to determine the initial velocities and also indicates the formation of a product inhibitor, probably resorufin sulfate ester, with Ki less than Km. Sensitivity of the reaction is less than 1 pmol/min. The maximal rate of resorufin sulfation by the bovine lung enzyme is estimated at 57 nmol/mg/min, which is 10% of the rate with an optimal substrate 2-naphthol. This assay may be most sensitive for phenol sulfotransferases with optimal activities at greater than pH 6, due to the acid-base properties of resorufin (pK alpha 6), which becomes nonfluorescent upon protonation.     

Bacterial expression and characterization of a cDNA for human liver estrogen sulfotransferase

A distinct human estrogen sulfotransferase (hEST-1) cDNA has been isolated from a human liver γZap cDNA library using a PCR procedure. The enzymatically active protein has been expressed in two bacterial expression systems and the kinetic and immunologic properties of the enzyme have been characterized. The full-length cDNA for hEST-1 is 994 base pairs in length and encodes a 294 amino acid protein with a calculated molecular mass of 35,123 Da. Purified hEST-1 migrated with an apparent molecular mass of 35,000 Da during SDS-polyacrylamide gel electrophoresis. Immunoblot analysis of hEST-1 expressed in E. coli with a rabbit anti-hEST-1 antibody yields a band of approximately 35,000 Da. The anti-hEST-1 antibody also detects a single band in human liver and jejunum cytosol which migrates with the same molecular mass as expressed hEST-1. There was also no cross-reactivity of hEST-1 with rabbit anti-hP-PST or rabbit anti-hDHEA-ST antibodies upon immunoblot analysis. hEST-1 was expressed in bacteria and purified to homogeneity. Expressed hEST-1 activity has a significantly greater affinity for estrogen sulfation than that found for the other human STs which conjugate estrogens. hEST-1 maximally sulfates β-estradiol and estrone at concentrations of 20 nM. hEST-1 also sulfates dehydroepiandrosterone, pregnenolone, ethinylestradiol, and 1-naphthol, at significantly higher concentrations; however, cortisol, testosterone and dopamine are not sulfated. The results presented in this paper describe the expression and characterization of a human EST distinct from other human STs which sulfate estrogens. The high affinity of hEST-1 for estrogens indicates that this ST may be important in both the metabolism of estrogens and in the regulation of their activities.     

Correlation between PAP-dependent steroid binding activity and substrate specificity of mouse and human estrogen sulfotransferases

Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the sulfoconjugation and inactivation of estrogens using 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as an activated sulfate donor. A finding of undetermined significance in the study of EST has been that the guinea pig EST is able to bind pregnenolone and estradiol with high affinity in the presence of PAP, the reaction by-product of the sulfate donor PAPS. This finding has raised the possibility that EST may have other physiological functions independent of its enzymatic activity as a sulfotransferase. To determine if the PAP-dependent steroid binding activity is a common property shared by other estrogen sulfotransferases, we have expressed the mouse and human EST in bacteria and used the purified protein to address this question. We found that, in the presence of PAP, both recombinant mouse and human EST were able to bind estradiol with high affinity but only the human EST was able to bind pregnenolone. In addition, we show that human but not the mouse EST was also able to bind dehydroepiandrosterone, a property that was not described for the guinea pig EST. Furthermore, we demonstrate that the promiscuity of human EST in steroid binding is mirrored by a correspondingly low substrate specificity in its enzymatic activity as a sulfotransferase. Reversely, the lack of stable binding of pregnenolone and dehydroepiandrosterone by the mouse EST is paralleled by a lack of sulfotransferase activity of this enzyme toward these two steroids. Mutagenesis of mouse EST within a domain critical for PAPS binding abolished both its sulfotransferase and PAP-dependent estrogen binding activity. These data suggest that stable binding of steroids such as pregnenolone or estrogen is not an independent property of estrogen sulfotransferases but rather is related to their catalytic activity.     

Tyrosine sulfation of statherin

Tyrosylprotein sulfotransferase (TPST), responsible for the sulfation of a variety of secretory and membrane proteins, has been identified and characterized in submandibular salivary glands (William et al. Arch Biochem Biophys 1997; 338: 90-96). In the present study we demonstrate the sulfation of a salivary secretory protein, statherin, by the tyrosylprotein sulfotransferase present in human saliva. Optimum statherin sulfation was observed at pH 6.5 and at 20 mm MnCl(2). Increase in the level of total sulfation was observed with increasing statherin concentration. The K(m)value of tyrosylprotein sulfotransferase for statherin was 40 microM. Analysis of the sulfated statherin product on SDS-polyacrylamide gel electrophoresis followed by autoradiography revealed (35)S-labelling of a 5 kDa statherin. Further analysis of the sulfated statherin revealed the sulfation on tyrosyl residue. This study is the first report demonstrating tyrosine sulfation of a salivary secretory protein. The implications of this sulfation of statherin in hydroxyapatite binding and Actinomyces viscosus interactions are discussed.     

Rat liver aryl sulfotransferase-catalyzed sulfation and rearrangement of 9-fluorenone oxime

The role of hepatic cytosolic aryl sulfotransferase (3'-phosphoadenylylsulfate:phenol sulfotransferase, EC 2.8.2.1) in the enzymic rearrangement of 9-fluorenone oxime to phenanthridone was investigated. 9-Fluorenone oxime was found to be an excellent substrate for a partially purified rat liver aryl sulfotransferase preparation. This compound was in fact superior to 2-naphthol, the standard assay substrate. This is the first reported observation of aryl oxime sulfation by the aryl sulfotransferases. 9-Fluorenone oxime sulfation exhibited pronounced substrate inhibition at high substrate concentrations. However, despite virtually complete conversion of 9-fluorenone oxime to the corresponding N-O-sulfate conjugate in enzyme incubation mixtures, only small amounts of rearrangement product were detected after long-term incubations. In addition, 9-fluorenone oxime-O-sulfonic acid was chemically synthesized and tested for stability. The results showed that rearrangement was pH-dependent and occurred slowly over several hours. It is therefore concluded that aryl sulfotransferase-catalyzed sulfation likely plays an important role in the in vitro and in vivo disposition of 9-fluorenone oxime. Moreover, sulfation facilitates the Beckmann-like conversion of 9-fluorenone oxime to phenanthridone. Sulfation alone, however, appears insufficient to account for all of the previously reported in vitro and in vivo rearrangement.     

Differential xenoestrogen-sulfating activities of the human cytosolic sulfotransferases: molecular cloning,expression, and purification of human SULT2B1a and SULT2B1b sulfotransferases

Environmental xenoestrogens have been implicated in human reproductive disorders and an increased incidence of breast cancer. Sulfation, a Phase II detoxification mechanism involving the cytosolic sulfotransferases (STs), may be an important mechanism in vivo for fending off these compounds. In this study, we report on the molecular cloning, expression, and purification of two human cytosolic STs, SULT2B1a and SULT2b1b. The activities of these two enzymes, as well as the other eight known human cytosolic STs previously prepared, toward representative environmental xenoestrogens were examined. Activity data showed that P-form (SULT1A1) PST displayed the highest activity toward these compounds, while SULT1C ST #2 also showed considerable activity, indicating that these enzymes may play a more important role in detoxification of environmental xenoestrogens. SULT1C ST #1, SULT2B1a ST, SULT2B1b ST and NST showed negligible or undetectable activity toward these compounds. The other four enzymes, M-form (SULT1A3) PST, SULT1B2 ST, SULT2A1 ST and SULT1E ST showed intermediate levels of activity toward some of these compounds. Kinetic studies on the sulfation of xenoestrogens by P-form (SULT1A1) PST were performed. The results are interpreted in the context of the endocrine-disrupting nature of these xenoestrogens.     

Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases

Sulfated glycans play critical roles during the development, differentiation and growth of various organisms. The most well-studied sulfated molecules are sulfated glycosaminoglycans (GAGs). Recent incidents of heparin drug contamination convey the importance of having a convenient and sensitive method for detecting different GAGs. Here, we describe a molecular method to detect GAGs in biological and biomedical samples. Because the sulfation of GAGs is generally not saturated in vivo, it is possible to introduce the radioisotope (35)S in vitro using recombinant sulfotransferases, thereby allowing detection of minute quantities of these molecules. This strategy was also successfully applied in the detection of other glycans. As examples, we detected contaminant GAGs in commercial heparin, heparan sulfate and chondroitin samples. The identities of the contaminant GAGs were further confirmed by lyase digestion. Oversulfated chondroitin sulfate was detectable only following a simple desulfation step. Additionally, in vitro sulfation by sulfotransferases allowed us to map glycan epitopes in biological samples. This was illustrated using mouse embryo and rat organ tissue sections labeled with the following carbohydrate sulfotransferases: CHST3, CHST15, HS3ST1, CHST4 and CHST10.     

Differential xenoestrogen-sulfating activities of the human cytosolic sulfotransferases: molecular cloning,expression, and purification of human SULT2B1a and SULT2B1b sulfotransferases

Environmental xenoestrogens have been implicated in human reproductive disorders and an increased incidence of breast cancer. Sulfation, a Phase II detoxification mechanism involving the cytosolic sulfotransferases (STs), may be an important mechanism in vivo for fending off these compounds. In this study, we report on the molecular cloning, expression, and purification of two human cytosolic STs, SULT2B1a and SULT2b1b. The activities of these two enzymes, as well as the other eight known human cytosolic STs previously prepared, toward representative environmental xenoestrogens were examined. Activity data showed that P-form (SULT1A1) PST displayed the highest activity toward these compounds, while SULT1C ST #2 also showed considerable activity, indicating that these enzymes may play a more important role in detoxification of environmental xenoestrogens. SULT1C ST #1, SULT2B1a ST, SULT2B1b ST and NST showed negligible or undetectable activity toward these compounds. The other four enzymes, M-form (SULT1A3) PST, SULT1B2 ST, SULT2A1 ST and SULT1E ST showed intermediate levels of activity toward some of these compounds. Kinetic studies on the sulfation of xenoestrogens by P-form (SULT1A1) PST were performed. The results are interpreted in the context of the endocrine-disrupting nature of these xenoestrogens.     

Sulfation of ractopamine and salbutamol by the human cytosolic sulfotransferases

Feed additives such as ractopamine and salbutamol are pharmacologically active compounds, acting primarily as β-adrenergic agonists. This study was designed to investigate whether the sulfation of ractopamine and salbutamol may occur under the metabolic conditions and to identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating two major feed additive compounds, ractopamine and salbutamol. A metabolic labelling study showed the generation and release of [(35)S]sulfated ractopamine and salbutamol by HepG2 human hepatoma cells labelled with [(35)S]sulfate in the presence of these two compounds. A systematic analysis using 11 purified human SULTs revealed SULT1A3 as the major SULT responsible for the sulfation of ractopamine and salbutamol. The pH dependence and kinetic parameters were analyzed. Moreover, the inhibitory effects of ractopamine and salbutamol on SULT1A3-mediated dopamine sulfation were investigated. Cytosol or S9 fractions of human lung, liver, kidney and small intestine were examined to verify the presence of ractopamine-/salbutamol-sulfating activity in vivo. Of the four human organs, the small intestine displayed the highest activity towards both compounds. Collectively, these results imply that the sulfation mediated by SULT1A3 may play an important role in the metabolism and detoxification of ractopamine and salbutamol.     

Manganese-dependent Dopa/tyrosine sulfation in HepG2 human hepatoma cells: novel Dopa/tyrosine sulfotransferase activities associated with the human monoamine-form phenol sulfotransferase

Human monoamine (M)-form phenol sulfotransferase (PST) was PCR-cloned and transiently expressed in COS-7 cells. The recombinant enzyme was demonstrated to display not only the previously reported sulfotransferase activity toward dopamine, but also novel manganese-dependent Dopa/tyrosine sulfotransferase activities. These results imply a new functional role of the human M-form PST in the homeostatic regulation of Dopa and tyrosine.     

4-Hydroxytamoxifen sulfation metabolism

Tamoxifen (TAM) is an important chemotherapeutic agent for the treatment of breast cancer. It has also been shown to decrease breast cancer incidence in healthy women at high risk for the disease. The increased risk of endometrial cancer in women has raised concerns in the use of the drug. Tamoxifen has also been shown to be a potent hepatocarcinogen in rats. The oxidative metabolites of TAM include alpha-hydroxytamoxifen (alpha-OH-TAM) and 4-hydroxytamoxifen (4-OH-TAM). The studies on the sulfation of these metabolites are very limited. It has been reported that alpha-OH-TAM is a substrate for rat hydroxysteroid sulfotransferase a (STa). Our studies on the sulfation of 4-OH-TAM demonstrated that 4-hydroxytamoxifen can be sulfated by human liver and human intestinal cytosols. Human phenol-sulfating sulfotransferase and human estrogen sulfotransferase are the major enzymes for the sulfation of 4-OH-TAM. Human dopamine-sulfating sulfotransferase also has sulfation activity for 4-OH-TAM. In contrast, rat liver and intestine cytosols have no detectable sulfation activity for 4-OH-TAM. The results suggest that the alpha-OH-TAM sulfation pathway leads to bioactivation of TAM, and the 4-OH-TAM sulfation pathway leads to detoxification of TAM. This agrees with the fact that TAM is more toxic for rats than for human beings.