Charles N. Falany

Enzymology of human cytosolic sulfotransferases.

Conjugation of many xenobiotics, drugs, and endogenous compounds with a sulfonate moiety is an important reaction in their biotransformation. Sulfation of these compounds generally results in a decrease in biological activity and an increase in their urinary excretion. However, in certain instances, sulfation results in bioactivation to reactive electrophilic or therapeutically active forms. At least four cytosolic sulfotransferases (STs) have been identified and characterized from human tissues. These enzymes are two forms of phenol ST (PST), the phenol‐sulfating and the monoamine‐sulfating forms of PST (P‐PST and M‐PST, respectively), an estrogen sulfotransferase (EST), and a hydroxysteroid ST, dehydroepiandrosterone ST (DHEA‐ST). Although four cytosolic STs have been well characterized in human tissues, evidence is accumulating for the presence of allelic forms or additional distinct forms of the STs in human tissues. The STs possess distinct but overlapping substrate specificities, and all of the STs are capable of conjugating both xenobiotic and endogenous compounds. The individual forms of ST may display distinct patterns of tissue specific expression and different mechanisms of regulation. Although the role of sulfation in drug metabolism is well recognized, an increased understanding of the biochemistry and molecular biology of the STs should also provide additional information as to their functions in many normal physiologic processes.—Falany, C. N. Enzymology of human cytosolic sulfotransferases. FASEB J. 11, 206‐216 (1997)

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 lambda Zap 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 beta-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.

Sulfuryl Transfer: The Catalytic Mechanism of Human Estrogen Sulfotransferase

Estrogen sulfotransferase (EST) catalyzes the transfer of the sulfuryl group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to 17β-estradiol (E2). The sulfation of E2 prevents it from binding to, and thereby activating, the estrogen receptor. The regulation of EST appears to be causally linked to tumorigenesis in the breast and endometrium. In this study, recombinant human EST is characterized, and the catalytic mechanism of the transfer reaction is investigated in ligand binding and initial rate experiments. The native enzyme is a dimer of 35-kDa subunits. The apparent equilibrium constant for transfer to E2 is (4.5 ± 0.2) × 103 at pH 6.3 andT = 25 ± 2 °C. Initial rate studies provide the kinetic constants for the reaction and suggest a sequential mechanism. E2 is a partial substrate inhibitor (K i = 80 ± 5 nm). The binding of two E2 per EST subunit suggests that the partial inhibition occurs through binding at an allosteric site. In addition to providing the dissociation constants for the ligand-enzyme complexes, binding studies demonstrate that each substrate binds independently to the enzyme and that both the E·PAP·E2S andE·PAP·E2 dead-end complexes form. These results strongly suggest a Random Bi Bi mechanism with two dead-end complexes.

Steroid sulfation by expressed human cytosolic sulfotransferases

The human cytosolic sulfotransferases (STs), dehydroepiandrosterone sulfotransferase (DHEA-ST) and the phenol-sulfating form of phenol sulfotransferase, (P-PST), have been expressed in bacteria and used to investigate the ability of the cloned enzymes to conjugate steroids and related compounds. DHEA-ST was capable of sulfating all of the 3-hydroxysteroids, testosterone and estrogens tested as substrates. The 3-hydroxysteroids, androsterone, epiandrosterone and androstenediol, were conjugated at 50-60% of the rate of DHEA. Of the steroids tested, P-PST was capable of conjugating only the estrogens. The catechol estrogens, 2-hydroxyestradiol, 4-hydroxyestradiol and 4-hydroxyestrone, and compounds with estrogenic activity such as 17 alpha-ethynyl-estradiol and trans-4-hydroxytamoxifen, were also tested as substrates. DHEA-ST showed little or no sulfation activity with these compounds; however, all of these compounds were sulfated by P-PST. These results indicate that the expressed human STs are valuable in analyzing the overlapping substrate specificities of these enzymes and that P-PST may have an important role in the metabolism of estrogens and estrogenic compounds in human tissues.

Triclosan as A substrate and inhibitor of 3'-phosphoadenosine 5'-phosphosulfate-sulfotransferase and udp-glucuronosyl transferase in human liver fractions

Triclosan is a broad spectrum antibacterial agent used in many household products. Due to its structural similarity to polychlorobiphenylols, which are potent inhibitors of the sulfonation and glucuronidation of 3-hydroxy-benzo[a]pyrene, it was hypothesized that triclosan would inhibit these phase II enzymes. This study was designed to assess the interactions of triclosan as a substrate and inhibitor of 3′-phosphoadenosine 5′-phosphosulfate-sulfotransferases and UDP-glucuronosyltransferases in human liver cytosol and microsomes. Triclosan was sulfonated and glucuronidated in human liver. The apparent Km and Vmax values for triclosan sulfonation were 8.5 μM and 0.096 nmol/min/mg protein, whereas Km and Vmax values for glucuronidation were 107 μM and 0.739 nmol/min/mg protein. Triclosan inhibited the hepatic cytosolic sulfonation of 3-hydroxybenzo(a)pyrene (3-OH-BaP), bisphenol A, p-nitrophenol, and acetaminophen with IC50 concentrations of 2.87, 2.96, 6.45, and 17.8 μM, respectively. Studies of 3-OH-BaP sulfonation by expressed human SULT1A1*1, SULT1A1*2, SULT1B1, and SULT1E1 showed that triclosan inhibited the activities of each of these purified enzymes with IC50 concentrations between 2.09 and 7.5 μM. Triclosan was generally a less potent inhibitor of microsomal glucuronidation. IC50 concentrations for triclosan with 3-OH-BaP, acetaminophen, and bisphenol A as substrates were 4.55, 297, and >200 μM, respectively. Morphine glucuronidation was not inhibited by 50 μM triclosan. The kinetics of 3-OH-BaP sulfonation and glucuronidation were examined in the presence of varying concentrations of triclosan: the inhibition of sulfonation was noncompetitive, whereas that of glucuronidation was competitive. These findings reveal that the commonly used bactericide triclosan is a selective inhibitor of the glucuronidation and sulfonation of phenolic xenobiotics.

Potent Inhibition of Estrogen Sulfotransferase by Hydroxylated Metabolites of Polyhalogenated Aromatic Hydrocarbons Reveals Alternative Mechanism for Estrogenic Activity of Endocrine Disrupters

Polyhalogenated aromatic hydrocarbons (PHAHs), such as polychlorinated dibenzo-p-dioxins and dibenzofurans, polybrominated diphenylethers, and bisphenol A derivatives are persistent environmental pollutants, which are capable of interfering with reproductive and endocrine function in birds, fish, reptiles, and mammals. PHAHs exert estrogenic effects that may be mediated in part by their hydroxylated metabolites (PHAH-OHs), the mechanisms of which remain to be identified. PHAH-OHs show low affinity for the ER. Alternatively, they may exert their estrogenic effects by inhibiting E2 metabolism. As sulfation of E2 by estrogen sulfotransferase (SULT1E1) is an important pathway for E2 inactivation, inhibition of SULT1E1 may lead to an increased bioavailability of estrogens in tissues expressing this enzyme. Therefore, we studied the possible inhibition of human SULT1E1 by hydroxylated PHAH metabolites and the sulfation of the different compounds by SULT1E1. We found marked inhibition of SULT1E1 by various PHAH-OHs, in particular by compounds with two adjacent halogen substituents around the hydroxyl group that were effective at (sub)nanomolar concentrations. Depending on the structure, the inhibition is primarily competitive or noncompetitive. Most PHAH-OHs are also sulfated by SULT1E1. We also investigated the inhibitory effects of the various PHAH-OHs on E2 sulfation by human liver cytosol and found that the effects were strongly correlated with their inhibitions of recombinant SULT1E1 (r = 0.922). Based on these results, we hypothesize that hydroxylated PHAHs exert their estrogenic effects at least in part by inhibiting SULT1E1-catalyzed E2 sulfation.

Sulfotransferases: genetics and role in toxicology

The mammalian xenobiotic-metabolizing sulfotransferases are cytosolic enzymes, which form a gene superfamily (SULT). Ten distinct human SULT forms are known. Two SULT forms represent splice variants, the other forms are encoded by separate genes. Common functional polymorphisms of the transcribed region are known for two of the forms. We have expressed 16 separate rat and human SULTs as well as some of their allelic variants, in Salmonella typhimurium TA1538 and/or V79 cells, which are target cells of commonly used mutagenicity assays. The expressed SULTs activated numerous compounds to mutagens in both assay systems. However, some promutagens were activated by only one or several of the human SULTs. Pronounced differences in promutagen activation were also detected between orthologous rat and human SULTs, and between allelic variants of human SULTs.

Pharmacogenetics of human cytosolic sulfotransferases

Cytosolic sulfotransferases (SULTs) are phase II detoxification enzymes that are involved in the biotransformation of a wide variety of structurally diverse endo- and xenobiotics, including many therapeutic agents and endogenous steroids. Single-nucleotide polymorphisms (SNPs) in SULTs have functional consequences on the translated protein. For the most part, these SNPs are fairly uncommon in the population, but some, most notably for SULT isoform 1A1, are commonly found and have been associated with cancer risk for a variety of tumor sites and also with response to therapeutic agents. SNPs in the hydroxysteroid sulfotransferase, SULT2A1, have been identified in African-American subjects and influence the ratio of plasma DHEA:DHEA-S. This modification could potentially influence cancer risk in steroidogenic tissues. SNPs in many SULTs are ethnically distributed, another factor that could influence SULT pharmacogenetics. Finally, genetic variation has also been identified in 3′-phosphoadenoside 5′-phosphosulfate synthetase (PAPPS), the enzymes responsible for producing the obligatory cosubstrate for all sulfotransferases. Taken together, this variability could substantially influence the disposition of drugs metabolized by SULTs. Elucidation of the basis and effect of variability in sulfation could greatly impact individualized therapy in the future.

Expression and characterization of the human 3β-hydroxysteroid sulfotransferases (SULT2B1a and SULT2B1b)

The human hydroxysteroid sulfotransferase, dehydroepiandrosterone sulfotransferase (DHEA-ST), is highly expressed in liver and adrenal cortex and displays reactivity towards a broad range of hydroxysteroids including 3 beta-hydroxysteroids, 3 alpha-hydroxysteroids, estrogens with a 3-phenolic moiety, and 17-hydroxyl group of androgens. In contrast, characterization of the newly described human hydroxysteroid sulfotransferase SULT2B1 isoforms shows that these enzymes are selective for the sulfation of 3 beta-hydroxysteroids, such as pregnenolone, epiandrosterone, DHEA, and androstenediol. There was no activity detected towards testosterone, dexamethasone, beta-estradiol, androsterone, or p-nitrophenol. The SULT2B1 gene encodes two isoforms, SULT2B1a and SULT2B1b, which are generated by alternate splicing of the first exon; therefore the SULT2B1 isoforms differ at their N-terminals. Northern Blot analysis detected a SULT2B1 message in RNA isolated from the human prostate and placenta. No SULT2B1 message was observed in RNA isolated from human liver, colon, lung, kidney, brain, or testis tissue. Purified SULT2B1a was used to generate a specific rabbit polyclonal anti-SULT2B1 antibody. The anti-SULT2B1 antibody did not react with expressed human EST, P-PST-1, M-PST, DHEA-ST, or ST1B2, during immunoblot analysis. The substrate specificity of the expressed SULT2B1 isoforms suggests that these enzymes are capable of regulating the activity of adrenal androgens in human tissues via their inactivation by sulfation.

DEVELOPMENTAL EXPRESSION OF ARYL, ESTROGEN AND HYDROXYSTEROID SULFOTRANSFERASES IN PRE- AND POSTNATAL HUMAN LIVER

Aryl- (SULT1A1), estrogen- (SULT1E1), and hydroxysteroid- (SULT2A1) sulfotransferases (SULTs) are active determinants of xenobiotic detoxication and hormone metabolism in the adult human liver. To investigate the role of these conjugating enzymes in the developing human liver, the ontogeny of immunoreactive SULT1A1, SULT1E1, and SULT2A1 expression was characterized in a series of 235 pre- and postnatal human liver cytosols ranging in age from early gestation to a postnatal age of 18 years. Interindividual variability in expression levels was apparent for all three SULTs in pre- and postnatal liver samples. Expression of the three SULTs displayed distinctly different developmental profiles. Semiquantitative Western blot analyses indicated that SULT1A1 and SULT2A1 immunoreactive protein levels were readily detectable in the majority of developmental human liver cytosols throughout the prenatal period. Whereas SULT1A1 expression did not differ significantly among the various developmental stages, SULT2A1 expression increased during the third trimester of gestation and continued to increase during postnatal life. By contrast, SULT1E1, a cardinal estrogen-inactivating enzyme, achieved the highest levels of expression during the earliest periods of gestation in prenatal male livers, indicating a requisite role for estrogen inactivation in the developing male. The present analysis suggests that divergent regulatory mechanisms are responsible for the differential patterns of hepatic SULT1A1, SULT1E1, and SULT2A1 immunoreactive protein levels that occur during pre- and postnatal human development, and implicates a major role for sulfotransferase expression in the developing fetus.