Michael W. Duffel

[24] Aryl sulfotransferases

Publisher Summary Aryl Sulfotransferases are a group of enzymes that are active with a wide variety of phenolic compounds as substrates. The aryl sulfotransferases fall into two families. The first family consists of aryl sulfotransferases I and II. Second family comprises of aryl sulfotransferases III and IV, which also resemble each other in their specificity for phenols. This chapter presents a procedure for the preparation and purification of these two families of aryl sulfotransferases. In preparation, the assay takes advantage of the partition into an organic solvent of the ion pair formed between certain organic sulfate monoesters and methylene blue. 2-Naphthol and adenosine 3-phosphate 5-phosphosulfate serve as substrates. The purification of all four aryl sulfotransferases is conducted together until the stage of partial separation upon elution from diethylaminoethyl cellulosexa0 /cellulose. The procedure has been applied to about 50 rat livers (0.5 kg) obtained from well-fed, male Sprague–Dawley TM rats weighing about 200 g. Livers are maintained at -80 ° prior to use.

Hydroxylated polychlorinated biphenyls are substrates and inhibitors of human hydroxysteroid sulfotransferase SULT2A1

Polychlorinated biphenyls (PCBs) are important persistent environmental contaminants. PCBs can be metabolically converted to their hydroxylated metabolites (OHPCBs), and in recent years, these OHPCBs have been observed to inhibit human sulfotransferases (SULTs) such as the phenol SULTs (SULT family-1) involved in the metabolism of estrogen and various other endogenous and xenobiotic phenols. In the present study, we have investigated the hypothesis that OHPCBs interact with family 2 hydroxysteroid (alcohol) SULTs (e.g., human SULT2A1), enzymes that are physiologically important for the metabolic transformations of several key endogenous hydroxysteroids as well as xenobiotic alcohols. We have examined the interactions of three OHPCBs with purified recombinant human SULT2A1 (also known as either human DHEA-ST or ST2A3). These studies with SULT2A1 were carried out on 4-hydroxy-2,5-dichlorobiphenyl (4-OH PCB 9), 4-hydroxy-2,3,5-trichlorobiphenyl (4-OH PCB 34), and 4-hydroxy-2,3,4,5-tetrachlorobiphenyl (4-OH PCB 68). Our results showed that 4-OH PCB 34 and 4-OH PCB 68 were substrates for SULT2A1, and 4-OH PCB 34 exhibited substrate inhibition similar to that seen with the physiological substrate dehydroepiandrosterone (DHEA). Although the sulfation of 4-OH PCB 34 and 4-OH PCB 68 represents a potential metabolic route for these compounds, these OHPCBs may also compete with other xenobiotic substrates as well as endogenous substrates for SULT2A1. The third OHPCB studied, 4-OH PCB 9, was not a substrate for SULT2A1 but was an inhibitor of the enzyme. Thus, the interactions of OHPCBs with human SULT2A1 represent both a potential route of metabolism and a possible source of interference with sulfation reactions catalyzed by this enzyme.

ENZYMATIC ASPECTS OF THE PHENOL (ARYL) SULFOTRANSFERASES

The sulfotransferases that are active in the metabolism of xenobiotics represent a large family of enzymes that catalyze the transfer of the sulfuryl group from 3′-phosphoadenosine 5′-phosphosulfate to phenols, to primary and secondary alcohols, to several additional oxygen-containing functional groups, and to amines. Restriction of this review to the catalytic processes of phenol or aryl sulfotransferases does not really narrow the field, because these enzymes have overlapping specificity, not only for specific compounds, but also for multiple functional groups. The presentation aims to provide an overview of the wealth of phenol sulfotransferases that are available for study but concentrates on the enzymology of rat and human enzymes, particularly on the predominant phenol sulfotransferase from rat liver. The kinetics and catalytic mechanism of the rat enzyme is extensively reviewed and is compared with observations from other sulfotransferases.

Tyrosine-ester sulfotransferase from rat liver: Bacterial expression and identification

A nucleotide sequence that had been proposed for, but not identified as, rat liver aryl sulfotransferase (EC 2.8.2.1) was prepared in an appropriate vector and transformed into Escherichia coli. The protein, expressed in large amounts, was not aryl sulfotransferase (EC 2.8.2.1) but rather tyrosine-ester sulfotransferase (EC 2.8.2.9), a sulfotransferase also active with phenols but having a much wider substrate range that includes hydroxylamines and esters of tyrosine. The recombinant tyrosine-ester sulfotransferase was identified by its unique substrate spectrum, by comparison with three peptides that were sequenced from homogeneous tyrosine-ester sulfotransferase isolated directly from rat liver, and by the specificity of antibody raised to the rat liver enzyme. Two isoforms were obtained, each of which was difficult to solubilize upon sonication of E. coli. Both forms were solubilized with a solution of polyols (glycerol and sucrose) and subsequently purified to homogeneity.

Identification of Sulfated Metabolites of 4-Chlorobiphenyl (PCB3) in the Serum and Urine of Male Rats

Polychlorinated biphenyls (PCBs) are legacy pollutants that exert toxicities through various mechanisms. In recent years exposure to PCBs via inhalation has been recognized as a hazard. Those PCBs with lower numbers of chlorine atoms (LC-PCBs) are semivolatile and have been reported in urban air, as well as in the indoor air of older buildings. LC-PCBs are bioactivated to phenols and further to quinone electrophiles with genotoxic/carcinogenic potential. We hypothesized that phenolic LC-PCBs are subject to conjugation and excretion in the urine. PCB3, often present in high concentrations in air, is a prototypical congener for the study of the metabolism and toxicity of LC-PCBs. Our objective was to identify metabolites of PCB3 in urine that could be potentially employed in the estimation of exposure to LC-PCBs. Male Sprague-Dawley rats (150-175 g) were housed in metabolism cages and received a single intraperitoneal injection of 600 μmol/kg body weight of PCB3. Urine was collected every 4 h; rats were euthanized at 36 h; and serum was collected. LC/MS analysis of urine before and after incubation with β-glucuronidase and sulfatase showed that sulfate conjugates were in higher concentrations than glucuronide conjugates and free phenolic forms. At least two major metabolites and two minor metabolites were identified in urine that could be attributed to mercapturic acid metabolites of PCB3. Quantitation by authentic standards confirmed that approximately 3% of the dose was excreted in the urine as sulfates over 36 h, with peak excretion occurring at 10-20 h after exposure. The major metabolites were 4PCB3sulfate, 3PCB3 sulfate, 2PCB3 sulfate, and presumably a catechol sulfate. The serum concentration of 4PCB3 sulfate was 6.18 ± 2.16 μg/mL. This is the first report that sulfated metabolites of PCBs are formed in vivo. These findings suggest a prospective approach for exposure assessment of LC-PCBs by analysis of phase II metabolites in urine.

Structure-activity relationships for hydroxylated polychlorinated biphenyls as inhibitors of the sulfation of dehydroepiandrosterone catalyzed by human hydroxysteroid sulfotransferase SULT2A1.

Polychlorinated biphenyls (PCBs) are persistent worldwide pollutants that are of concern due to their bioaccumulation and health effects. Metabolic oxidation of PCBs results in the formation of hydroxylated metabolites (OHPCBs). Among their biological effects, OHPCBs have been shown to alter the metabolism of endocrine hormones, including inhibition of mammalian cytosolic sulfotransferases (SULTs) that are responsible for the inactivation of thyroid hormones and phenolic steroids (i.e., hSULT1A1, hSULT1B1, and hSULT1E1). OHPCBs also interact with a human hydroxysteroid sulfotransferase that plays a role in the sulfation of endogenous alcohol-containing steroid hormones and bile acids (i.e., hSULT2A1). The objectives of our current study were to examine the effects of a series of OHPCB congeners on the activity of hSULT2A1 and to develop a three-dimensional quantitative structure-activity relationship (3D-QSAR) model for OHPCBs as inhibitors of the enzyme. A total of 15 OHPCBs were examined, and the sulfation of 1 μM [(3)H] dehydroepiandrosterone (DHEA) was utilized as a model reaction catalyzed by the enzyme. All 15 OHPCBs inhibited the sulfation of DHEA, with IC(50) values ranging from 0.6 μM to 96 μM, and eight of these OHPCBs were also substrates for the enzyme. Comparative molecular field analysis (CoMFA) provided a predictive 3D-QSAR model with a q(2) value of 0.697 and an r(2) value of 0.949. The OHPCBs that had the highest potency as inhibitors of DHEA sulfation were those with a 3, 5-dichloro-4-hydroxy substitution pattern on the biphenyl ring system, and these congeners were also substrates for sulfation catalyzed by hSULT2A1.

Assay of purified aryl sulfotransferase suitable for reactions yielding unstable sulfuric acid esters

An assay procedure for purified aryl sulfotransferase is described. The method utilizes isocratic paired-ion reverse-phase HPLC analysis of adenosine-3,5-diphosphate formed in the reaction. Evaluation of the assay procedure was carried out with 1-naphthalene-methanol as a model substrate for purified rat hepatic aryl sulfotransferase IV. Kinetic constants for sulfation of 1-naphthalenemethanol determined by this method compared favorably with those determined using thin-layer chromatographic assays of 35S incorporation. These results indicate that the method will be suitable for determination of kinetic constants in sulfotransferase-catalyzed reactions where the product sulfuric acid ester may be chemically unstable.

Sulfated Metabolites of Polychlorinated Biphenyls Are High-Affinity Ligands for the Thyroid Hormone Transport Protein Transthyretin

Background: The displacement of l-thyroxine (T4) from binding sites on transthyretin (TTR) is considered a significant contributing mechanism in polychlorinated biphenyl (PCB)-induced thyroid disruption. Previous research has discovered hydroxylated PCB metabolites (OH-PCBs) as high-affinity ligands for TTR, but the binding potential of conjugated PCB metabolites such as PCB sulfates has not been explored. Objectives: We evaluated the binding of five lower-chlorinated PCB sulfates to human TTR and compared their binding characteristics to those determined for their OH-PCB precursors and for T4. Methods: We used fluorescence probe displacement studies and molecular docking simulations to characterize the binding of PCB sulfates to TTR. The stability of PCB sulfates and the reversibility of these interactions were characterized by HPLC analysis of PCB sulfates after their binding to TTR. The ability of OH-PCBs to serve as substrates for human cytosolic sulfotransferase 1A1 (hSULT1A1) was assessed by OH-PCB–dependent formation of adenosine-3´,5´-diphosphate, an end product of the sulfation reaction. Results: All five PCB sulfates were able to bind to the high-affinity binding site of TTR with equilibrium dissociation constants (Kd values) in the low nanomolar range (4.8–16.8 nM), similar to that observed for T4 (4.7 nM). Docking simulations provided corroborating evidence for these binding interactions and indicated multiple high-affinity modes of binding. All OH-PCB precursors for these sulfates were found to be substrates for hSULT1A1. Conclusions: Our findings show that PCB sulfates are high-affinity ligands for human TTR and therefore indicate, for the first time, a potential relevance for these metabolites in PCB-induced thyroid disruption.

Substrate inhibition in human hydroxysteroid sulfotransferase SULT2A1: Studies on the formation of catalytically non-productive enzyme complexes

The cytosolic sulfotransferase hSULT2A1 is the major hydroxysteroid (alcohol) sulfotransferase in human liver, and it catalyzes the 3-phosphoadenosine-5-phosphosulfate (PAPS)-dependent sulfation of various endogenous hydroxysteroids as well as many xenobiotics that contain alcohol and phenol functional groups. The hSULT2A1 often displays substrate inhibition, and we have hypothesized that a key element in this response to increasing substrate concentration is the formation of non-productive ternary dead-end enzyme complexes involving the nucleotide product, adenosine 3,5-diphosphate (PAP). One of these substrates for hSULT2A1 is dehydroepiandrosterone (DHEA), a major circulating steroid hormone in humans that serves as precursor to both androgens and estrogens. We have utilized DHEA in both initial velocity studies and equilibrium binding experiments in order to evaluate the potential role of ternary complexes in substrate inhibition of the enzyme. Our results indicate that hSULT2A1 forms non-productive ternary complexes that involve either DHEA or dehydroepiandrosterone sulfate, and the formation of these ternary complexes displays negative cooperativity in the binding of DHEA.

2,2',3,3',6,6'-Hexachlorobiphenyl (PCB 136) is enantioselectively oxidized to hydroxylated metabolites by rat liver microsomes.

Developmental exposure to multiple ortho-substituted polychlorinated biphenyls (PCBs) causes adverse neurodevelopmental outcomes in laboratory animals and humans by mechanisms involving the sensitization of Ryanodine receptors (RyRs). In the case of PCB 136, the sensitization of RyR is enantiospecific, with only (-)-PCB 136 being active. However, the role of enantioselective metabolism in the developmental neurotoxicity of PCB 136 is poorly understood. The present study employed hepatic microsomes from phenobarbital (PB)-, dexamethasone (DEX)- and corn oil (VEH)-treated male Sprague-Dawley rats to investigate the hypothesis that PCB 136 atropisomers are enantioselectively metabolized by P450 enzymes to potentially neurotoxic, hydroxylated PCB 136 metabolites. The results demonstrated the time- and isoform-dependent formation of three metabolites, with 5-OH-PCB 136 (2,2,3,3,6,6-hexachlorobiphenyl-5-ol) being the major metabolite. The formation of 5-OH-PCB 136 increased with the activity of P450 2B enzymes in the microsomal preparation, which is consistent with PCB 136 metabolism by rat P450 2B1. The minor metabolite 4-OH-PCB 136 (2,2,3,3,6,6-hexachlorobiphenyl-4-ol) was produced by a currently unidentified P450 enzyme. An enantiomeric enrichment of (-)-PCB 136 was observed in microsomal incubations due to the preferential metabolism of (+)-PCB 136 to the corresponding 5-OH-PCB 136 atropisomer. 4-OH-PCB 136 displayed an enrichment of the atropisomer formed from (-)-PCB 136; however, the enrichment of this metabolite atropisomer did not affect the enantiomeric enrichment of the parent PCB because 4-OH-PCB 136 is only a minor metabolite. Although the formation of 5- and 4-OH-PCB 136 atropisomers increased with time, the enantioselective formation of the OH-PCB metabolites resulted in constant enantiomeric enrichment, especially at later incubation times. These observations not only demonstrate that the chiral signatures of PCBs and their metabolites in wildlife and humans are due to metabolism by P450 enzymes but also suggest that the enantioselective formation of neurotoxic PCB 136 metabolites, such as 4-OH-PCB 136, may play a role in the developmental neurotoxicity of PCBs.