The NSAID allosteric site of human cytosolic sulfotransferases.

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most commonly prescribed drugs worldwide-more than 111 million prescriptions were written in the United States in 2014. NSAIDs allosterically inhibit cytosolic sulfotransferases (SULTs) with high specificity and therapeutically relevant affinities. This study focuses on the interactions of SULT1A1 and mefenamic acid (MEF)-a potent, highly specific NSAID inhibitor of 1A1. Here, the first structure of an NSAID allosteric site-the MEF-binding site of SULT1A1-is determined using spin-label triangulation NMR. The structure is confirmed by site-directed mutagenesis and provides a molecular framework for understanding NSAID binding and isoform specificity. The mechanism of NSAID inhibition is explored using molecular dynamics and equilibrium and pre-steady-state ligand-binding studies. MEF inhibits SULT1A1 turnover through an indirect (helix-mediated) stabilization of the closed form of the active-site cap of the enzyme, which traps the nucleotide and slows its release. Using the NSAID-binding site structure of SULT1A1 as a comparative model, it appears that 11 of the 13 human SULT isoforms harbor an NSAID-binding site. We hypothesize that these sites evolved to enable SULT isoforms to respond to metabolites that lie within their metabolic domains. Finally, the NSAID-binding site structure offers a template for developing isozyme-specific allosteric inhibitors that can be used to regulate specific areas of sulfuryl-transfer metabolism.

Sulfotransferase-1A1-dependent bioactivation of aristolochic acid I and N-hydroxyaristolactam I in human cells.

Aristolochic acids (AA) are implicated in the development of chronic renal disease and upper urinary tract carcinoma in humans. Using in vitro approaches, we demonstrated that N-hydroxyaristolactams, metabolites derived from partial nitroreduction of AA, require sulfotransferase (SULT)-catalyzed conjugation with a sulfonyl group to form aristolactam-DNA adducts. Following up on this observation, bioactivation of AA-I and N-hydroxyaristolactam I (AL-I-NOH) was studied in human kidney (HK-2) and skin fibroblast (GM00637) cell lines. Pentachlorophenol, a known SULT inhibitor, significantly reduced cell death and aristolactam-DNA adduct levels in HK-2 cells following exposure to AA-I and AL-I-NOH, suggesting a role for Phase II metabolism in AA activation. A gene knockdown, siRNA approach was employed to establish the involvement of selected SULTs and nitroreductases in AA-I bioactivation. Silencing of SULT1A1 and PAPSS2 led to a significant decrease in aristolactam-DNA levels in both cell lines following exposure to AA-I, indicating the critical role for sulfonation in the activation of AA-I in vivo Since HK-2 cells proved relatively resistant to knockdown with siRNAs, gene silencing of xanthine oxidoreductase, cytochrome P450 oxidoreductase and NADPH:quinone oxidoreductase was conducted in GM00637 cells, showing a significant increase, decrease and no effect on aristolactam-DNA levels, respectively. In GM00637 cells exposed to AL-I-NOH, suppressing the SULT pathway led to a significant decrease in aristolactam-DNA formation, mirroring data obtained for AA-I. We conclude from these studies that SULT1A1 is involved in the bioactivation of AA-I through the sulfonation of AL-I-NOH, contributing significantly to the toxicities of AA observed in vivo.

The effects of endoxifen and other major metabolites of tamoxifen on the sulfation of estradiol catalyzed by human cytosolic sulfotransferases hSULT1E1 and hSULT1A1*1.

Tamoxifen is successfully used for both treatment and prevention of estrogen-dependent breast cancer, yet side effects and development of resistance remain problematic. Endoxifen is a major active metabolite of tamoxifen that is being investigated for clinical use. We hypothesized that endoxifen and perhaps other major metabolites of tamoxifen may affect the ability of human estrogen sulfotransferase 1E1 (hSULT1E1) and human phenol sulfotransferase 1A1 isoform 1 (hSULT1A1*1) to catalyze the sulfation of estradiol, an important mechanism in termination of estrogen signaling through loss of activity at estrogen receptors. Our results indicated that endoxifen, N-desmethyltamoxifen (N-desTAM), 4-hydroxytamoxifen (4-OHTAM), and tamoxifen-N-oxide were weak inhibitors of hSULT1E1 with Ki values ranging from 10 µM to 38 µM (i.e., over 1000 times higher than the 8.1 nM Km value for estradiol as substrate for the enzyme). In contrast to the results with hSULT1E1, endoxifen and 4-OHTAM were significant inhibitors of the sulfation of 2.0 µM estradiol catalyzed by hSULT1A1*1, with IC50 values (9.9 µM and 1.6 µM, respectively) that were similar to the Km value (1.5 µM) for estradiol as substrate for this enzyme. Additional investigation of the interaction of these metabolites with the two sulfotransferases revealed that endoxifen, 4-OHTAM, and N-desTAM were substrates for hSULT1E1 and hSULT1A1*1, although the relative catalytic efficiencies varied with both the substrate and the enzyme. These results may assist in future elucidation of cell- and tissue-specific effects of tamoxifen and its metabolites.

High accuracy in silico sulfotransferase models.

Predicting enzymatic behavior in silico is an integral part of our efforts to understand biology. Hundreds of millions of compounds lie in targeted in silico libraries waiting for their metabolic potential to be discovered. In silico "enzymes" capable of accurately determining whether compounds can inhibit or react is often the missing piece in this endeavor. This problem has now been solved for the cytosolic sulfotransferases (SULTs). SULTs regulate the bioactivities of thousands of compounds--endogenous metabolites, drugs and other xenobiotics--by transferring the sulfuryl moiety (SO3) from 3'-phosphoadenosine 5'-phosphosulfate to the hydroxyls and primary amines of these acceptors. SULT1A1 and 2A1 catalyze the majority of sulfation that occurs during human Phase II metabolism. Here, recent insights into the structure and dynamics of SULT binding and reactivity are incorporated into in silico models of 1A1 and 2A1 that are used to identify substrates and inhibitors in a structurally diverse set of 1,455 high value compounds: the FDA-approved small molecule drugs. The SULT1A1 models predict 76 substrates. Of these, 53 were known substrates. Of the remaining 23, 21 were tested, and all were sulfated. The SULT2A1 models predict 22 substrates, 14 of which are known substrates. Of the remaining 8, 4 were tested, and all are substrates. The models proved to be 100% accurate in identifying substrates and made no false predictions at Kd thresholds of 100 µM. In total, 23 "new" drug substrates were identified, and new linkages to drug inhibitors are predicted. It now appears to be possible to accurately predict Phase II sulfonation in silico.

Cytosolic sulfotransferase 1A3 is induced by dopamine and protects neuronal cells from dopamine toxicity: role of D1 receptor-N-methyl-D-aspartate receptor coupling.

Dopamine neurotoxicity is associated with several neurodegenerative diseases, and neurons utilize several mechanisms, including uptake and metabolism, to protect them from injury. Metabolism of dopamine involves three enzymes: monoamine oxidase, catechol O-methyltransferase, and sulfotransferase. In primates but not lower order animals, a sulfotransferase (SULT1A3) is present that can rapidly metabolize dopamine to dopamine sulfate. Here, we show that SULT1A3 and a closely related protein SULT1A1 are highly inducible by dopamine. This involves activation of the D1 and NMDA receptors. Both ERK1/2 phosphorylation and calcineurin activation are required for induction. Pharmacological agents that inhibited induction or siRNA targeting SULT1A3 significantly increased the susceptibility of cells to dopamine toxicity. Taken together, these results show that dopamine can induce its own metabolism and protect neuron-like cells from damage, suggesting that SULT1A3 activity may be a risk factor for dopamine-dependent neurodegenerative diseases.

Highly selective bioactivation of 1- and 2-hydroxy-3-methylcholanthrene to mutagens by individual human and other mammalian sulphotransferases expressed in Salmonella typhimurium.

The benzylic alcohols 1- and 2-hydroxy-3-methylcholanthrene (OH-MC) are major primary metabolites of the carcinogen 3-methylcholanthrene (MC). We investigated them for mutagenicity in TA1538-derived Salmonella typhimurium strains expressing mammalian sulphotransferases (SULTs). 1-OH-MC was efficiently activated by human (h) SULT1B1 (2400 revertants/nmol), weakly activated by hSULT1C3 and hSULT2A1 (2-9 revertants/nmol), but not activated by the other hSULTs studied (1A2, 1A3, 1C2 and 1E1). Mouse, rat and dog SULT1B1 activated 1-OH-MC (8-100 revertants/nmol) with much lower efficiency than their human orthologue. The other isomer, 2-OH-MC, was activated to a potent mutagen by hSULT1A1 (4000-5400 revertants/nmol), weakly activated by hSULT1A2 or hSULT2A1 (1-12 revertants/nmol), but not activated by the other hSULTs. In contrast to their human orthologue, mouse, rat and dog SULT1A1 did not appreciably activate 2-OH-MC (<1 to 6 revertants/nmol), either. Instead, mouse and rat SULT1B1, unlike their human and canine orthologues, demonstrated some activation of 2-OH-MC (15-100 revertants/nmol). Docking analyses indicated that 1- and 2-OH-MC might bind to the active site of hSULT1A1 and hSULT1B1, but only for (S)-2-OH-MC/hSULT1A1 and (R)-1-OH-MC/hSULT1B1 with an orientation suitable for catalysis. Indeed, 1- and 2-OH-MC were potent inhibitors of the hSULT1A1-mediated sulphation of acetaminophen [concentration inhibiting the enzyme activity by 50% (IC50) 15 and 13nM, respectively]. This inhibition was weak with mouse, rat and dog SULT1A1 (IC50 ≥ 4 µM). Inhibition of the SULT1B1 enzymes was moderate, strongest for 1-OH-MC/hSULT1B1. In conclusion, this study provides examples for high selectivity of bioactivation of promutagens by an individual form of human SULT and for pronounced differences in activation capacity between orthologous SULTs from different mammalian species. These characteristics make the detection and evaluation of such mutagens extremely difficult, in particular as the critical form may even differ for positional isomers, such as 1- and 2-OH-MC. Moreover, the species-dependent differences will complicate the verification of in vitro results in animal studies.

Potent inhibition of human sulfotransferase 1A1 by 17α-ethinylestradiol: role of 3'-phosphoadenosine 5'-phosphosulfate binding and structural rearrangements in regulating inhibition and activity.

Sulfotransferase (SULT) 1A1 is the major drug/xenobiotic-conjugating SULT isoform in human liver because of its broad substrate reactivity and high expression level. SULT1A1 sulfates estrogens with low micromolar K(m) values consistent with its affinity for sulfation of many small phenolic compounds. Binding studies showed the unexpected ability of 17α-ethinylestradiol (EE2) to bind and inhibit SULT1A1 activity toward p-nitrophenol and ß-naphthol at low nanomolar concentrations, whereas EE2 was not sulfated until significantly higher concentrations were reached. EE2 had a K(i) of 10 nM for inhibiting p-nitrophenol and ß-naphthol sulfation and inhibited 17ß-estradiol (E2) sulfation in intact human MCF-7 breast cancer cells with a K(i) of 19 nM. In contrast, the K(m) for EE2 sulfation by SULT1A1 was 700 nM. The K(d) for EE2 binding of pure SULT1A1 was 0.5 ± 0.15 µM; however, the K(d) for EE2 binding to the SULT1A1-PAP complex was >100-fold lower (4.3 ± 1.7 nM). The K(d) for E2 binding to SULT1A1 changed from 2.3 ± 0.9 to 1.2 ± 0.56 µM in the presence of PAP. Docking studies with E2 indicate that E2 binds in a competent orientation in the resolved structure of SULT1A1 in the both presence and absence of 3'-phosphoadenosine 5'-phosphosulfate (PAPS). However, EE2 binds in a catalytically competent orientation in the absence of PAPS but in a noncompetent orientation via formation of a charge interaction with Tyr108 if PAPS is bound first. In conclusion, EE2 is a potent inhibitor, but not a substrate, of SULT1A1 at low nanomolar concentrations, indicating the possibility of drug-drug interactions during contraceptive therapy.

Hypothesis: holiday sudden cardiac death: food and alcohol inhibition of SULT1A enzymes as a precipitant.

Sudden cardiac death is a significant health issue, causing millions of deaths worldwide annually. Studies have found that the likelihood of such death is higher in winter. Further studies identified that the highest likelihood occurs on Christmas Day and New Years Day, but not the interim period. Thanksgiving, Independence Day and the Islamic holiday Eid Al-Fitr also show significant increases in the rate of cardiac events or death. A number of mechanisms have been proposed, but none have satisfactorily explained the evidence. This article reviews the data supporting the existence of a holiday cardiac death phenomenon, the involvement of catecholamines and the normal modes of human catecholamine deactivation. Further evidence is reviewed that supports a hypothesized mechanism whereby critical SULT1A catecholamine deactivation enzymes can in some patients be inhibited by naturally-occurring phenols and polyphenols in foods and alcohols. If deactivation is inhibited by holiday consumption excesses, holiday stress or excitement could lead to a buildup of catecholamines that can cause fatal arrhythmias. Awareness of this mechanism could reduce deaths, both through doctor/patient education leading to a moderation in consumption and through the potential identification of patients with a predisposition to SULT1A inhibition. This hypothesis also raises parallels between sudden cardiac death in adults and Sudden Infant Death Syndrome (SIDS). The possible involvement of SULT1A inhibition in SIDS is discussed.

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. Overall, data are consistent with release of PAP from E-PAP and PAPS from E-PAPS contributing to rate-limitation in both reaction directions.

Crystal structures of SULT1A2 and SULT1A1 *3: insights into the substrate inhibition and the role of Tyr149 in SULT1A2.

The cytosolic sulfotransferases (SULTs) in vertebrates catalyze the sulfonation of endogenous thyroid/steroid hormones and catecholamine neurotransmitters, as well as a variety of xenobiotics, using 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfonate donor. In this study, we determined the structures of SULT1A2 and an allozyme of SULT1A1, SULT1A1 *3, bound with 3'-phosphoadenosine 5'-phosphate (PAP), at 2.4 and 2.3A resolution, respectively. The conformational differences between the two structures revealed a plastic substrate-binding pocket with two channels and a switch-like substrate selectivity residue Phe247, providing clearly a structural basis for the substrate inhibition. In SULT1A2, Tyr149 extends approximately 2.1A further to the inside of the substrate-binding pocket, compared with the corresponding His149 residue in SULT1A1 *3. Site-directed mutagenesis study showed that, compared with the wild-type SULT1A2, mutant Tyr149Phe SULT1A2 exhibited a 40 times higher K(m) and two times lower V(max) with p-nitrophenol as substrate. These latter data imply a significant role of Tyr149 in the catalytic mechanism of SULT1A2.

Sulfation of dietary flavonoids by human sulfotransferases.

Dietary flavonoids catechin, epicatechin, eriodictyol, and hesperetin were investigated as substrates and inhibitors of human sulfotransferases (hSULTs). Purified recombinant proteins and human intestine cytosol were used as enzyme sources. hSULT1A1 and hSULT1A3 as well as human intestine cytosol can catalyse the sulfation of the investigated flavonoids. Sulfation of catechin, epicatechin, eriodictyol, and hesperetin by recombinant hSULTs showed substrate inhibition at high flavonoid concentrations. Hesperetin and eriodictyol are potent inhibitors of purified hSULT1A1, hSULT1A3, hSULT1E1, and hSULT2A1. Catechin and epicatechin inhibited hSULT1A1 and hSULT1A3, but not hSULT1E1 and hSULT2A1. The sulfation efficacy and potency of inhibition is related to the C-ring structure of flavonoids. These results suggest that dietary flavonoids may regulate human SULT activity and, therefore, affect the regulation of hormones and neurotransmitters, detoxification of drugs, and the bioactivation of pro- carcinogens and pro-mutagens.

Isoform-specific therapeutic control of sulfonation in humans.

The activities of hundreds, perhaps thousands, of metabolites are regulated by human cytosolic sulfotransferases (SULTs) - a 13-member family of disease relevant enzymes that catalyze transfer of the sulfuryl moiety (-SO3) from PAPS (3'-phosphoadenosine 5'-phosphosulfonate) to the hydroxyls and amines of acceptors. SULTs harbor two independent allosteric sites, one of which, the focus of this work, binds non-steroidal anti-inflammatory drugs (NSAIDs). The structure of the first NSAID-binding site - that of SULT1A1 - was elucidated recently and homology modeling suggest that variants of the site are present in all SULT isoforms. The objective of the current study was to assess whether the NSAID-binding site can be used to regulate sulfuryl transfer in humans in an isoform specific manner. Mefenamic acid (Mef) is a potent (Ki 27 nM) NSAID-inhibitor of SULT1A1 - the predominant SULT isoform in small intestine and liver. Acetaminophen (APAP), a SULT1A1 specific substrate, is extensively sulfonated in humans. Dehydroepiandrosterone (DHEA) is specific for SULT2A1, which we show here is insensitive to Mef inhibition. APAP and DHEA sulfonates are readily quantified in urine and thus the effects of Mef on APAP and DHEA sulfonation could be studied non-invasively. Compounds were given orally in a single therapeutic dose to a healthy, adult male human with a typical APAP-metabolite profile. Mef profoundly decreased APAP sulfonation during first pass metabolism and substantially decreased systemic APAP sulfonation without influencing DHEA sulfonation; thus, it appears the NSAID site can be used to control sulfonation in humans in a SULT-isoform specific manner.

Phytoestrogens and xenoestrogens: the contribution of diet and environment to endocrine disruption.

Some endocrine disrupting compounds such as phthalates and phenols act non-genomically by inhibiting the sulfotransferase (SULT 1E1 and SULT 1A1) isoforms which inactivate estrogens by sulfonation. A range of environmental phenolic contaminants and dietary flavonoids was tested for inhibition of the human SULT 1A1, 1E1 and 2A1 isoforms. In particular, the plasticisers 4-n-octyl- and 4-n-nonyl-phenol inhibit SULT 1E1 with IC(50) values of 0.16 microM vs. 10nM estradiol while the 2-substituted chlorophenols show similar values. Flavonoids are also SULT inhibitors; tricin is a competitive inhibitor of SULT 1E1 with a K(i) of 1.5+/-0.8 nM. In a small pilot study to determine whether ingestion of soy flavonoids would affect SULT1A1 activity in vivo as well as in vitro, sulfonation of daidzein was reduced in a group of women 'at risk' of breast cancer, as compared with controls, although the SULT 1A1*1/SULT 1A1*2 allele ratio was not different. Endocrine disrupting effects in man may be multifactorial when components from both the diet and the environment act at the same point in steroid metabolism.

Mutagenicity of N-nitrosodiethanolamine in a V79-derived cell line expressing two human biotransformation enzymes.

N-nitrosodiethanolamine (NDELA) has demonstrated carcinogenic activity in various rodent models. However, it is negative or only weakly active in standard in vitro genotoxicity assays. This poor response might be due to the requirement of specific enzymes for its activation. Previous work indicated that cytochrome P450 (CYP) 2E1, alcohol dehydrogenases and sulphotransferases (SULTs) can convert NDELA into reactive metabolites. We report here that NDELA induces concentration-dependent gene mutations (at the hprt locus) in V79-hCYP2E1-hSULT1A1 cells, engineered for expression of human CYP2E1 and human SULT1A1, but is inactive in parental V79 cells. Mutagenicity of NDELA in V79-hCYP2E1-hSULT1A1 cells was abolished by the CYP2E1 inhibitor 1-aminobenzotriazole, but unaffected by the SULT1A1 inhibitor pentachlorophenol. The efficiency and specificity of these inhibitors was demonstrated in gene mutation assays using SULT- and CYP2E1-dependent reference mutagens, 2-nitropropane and N-nitrosodimethylamine, respectively. In this study, it is documented for the first time that NDELA can induce gene mutations in mammalian cells. Whereas human CYP2E1 was required for its activation, human SULT1A1 was not involved either in its activation or its inactivation in our cell model.

Inhibition of human phenol and estrogen sulfotransferase by certain non-steroidal anti-inflammatory agents.

We hypothesized that aryl acetate- and aryl carboxylate-containing drugs would inhibit human phenol sulfotransferase (SULT1A1), and that selectivity would depend upon the interaction of the aryl portion of the molecule with the sulfotransferase acceptor binding site. This hypothesis was based on results with the rat orthologue showing that oxidation of phenolic substrates to carboxylate derivatives resulted in competitive inhibition of rat phenol sulfotransferase. We chose nine structurally representative non-steroidal anti-inflammatory agents and determined their inhibitory potency and selectivity toward SULT1A1 and expressed human estrogen sulfotransferase (SULT1E1). The results show that the tested agents reversibly inhibit SULT1A1 activity with IC(50) ranging from 0.1 microM to 3800 microM. These agents also inhibited SULT1E1 (IC(50) = 6 microM to 9000 microM). The agents were clearly isoform selective, with IC(50) ratios (1E1/1A1) ranging from 0.01 to 200. Nimesulide, meclofenamate, and piroxicam were more selective towards SULT1A1 inhibition, while sulindac and ibuprofen were more selective towards SULT1E1 inhibition. Sulfotransferase inhibition was maintained after substituting the carboxylate with enolate (nimesulide) or methylsulfonamide (piroxicam). Kinetic studies determined the type of inhibition of SULT1A1 for three agents (meclofenamate, nimesulide, aspirin) to be non-competitive or partial non-competitive versus both substrate (p-nitrophenol) and cofactor (PAPS). This inhibition mechanism indicates that meclofenamate, nimesulide and aspirin bind near enough to the substrate binding site to prevent catalysis but not affect dissociation of the substrate-enzyme complex. The inhibition of SULT1A1 by meclofenamate, nimesulide, salicylate and aspirin may be clinically relevant based on ratio of inhibition constant to predicted in vivo inhibitor concentration ([I]/IC(50) > 1).

Polychlorobiphenylols are selective inhibitors of human phenol sulfotransferase 1A1 with 4-nitrophenol as a substrate.

Polychlorobiphenylols (OH-PCBs) were reported as potent inhibitors of estrogen sulfotransferase, thyroid hormone and 3-hydroxybenzo(a)pyrene sulfotransferases. The aim of this study was to examine the effects of selected OH-PCBs on SULT1A1 activity in human liver cytosol, measured with 4microM 4-nitrophenol, a concentration considered to be diagnostic for selectively detecting SULT1A1. All the OH-PCBs studied inhibited the sulfonation of 4-nitrophenol in human liver cytosol. Among the eighteen OH-PCBs studied, 3'-OH-CB3 (4-chlorobiphenyl-3'-ol) was the most potent inhibitor (IC(50): 0.73+/-0.15microM, mean+/-S.D., n=3). The least potent inhibitor studied was 6'-OH-CB35 (3,3',4-trichlorobiphenyl-6'-ol) with IC(50): 49.1+/-10.8microM. The IC(50) values of the other OH-PCBs studied ranged from 0.78 to 3.76microM. Some OH-PCBs with various inhibitory potencies with human liver cytosol were selected for study with recombinant human SULT1A1 and SULT1B1. These OH-PCBs showed more potent inhibition of 4-nitrophenol sulfonation with SULT1A1 than with human liver cytosol. The IC(50) values with human liver cytosol showed a perfect linear correlation with those found with SULT1A1 (r(2)=1), but not with SULT1B1 (r(2)=0.21). The results suggested that in these human samples SULT1A1 was predominantly responsible for the sulfonation of 4-nitrophenol, with very little or no contribution from SULT1B1. The kinetics of inhibition were studied with 4'-OH-CB165, which is similar in structure to OH-PCBs found in human blood. The 4'-OH-CB165 was a mixed noncompetitive-uncompetitive inhibitor (K(i)=1.80+/-0.2microM, K(ies)=0.16+/-0.02microM). Finally, it was demonstrated that the tested OH-PCBs were themselves only slowly sulfonated by human sulfotransferases in the presence of (35)S-PAPS, as measured by the production of (35)S-labeled metabolites. Although this series of 18 OH-PCBs was too small to draw conclusions about structure-potency relationships, this work demonstrated that several OH-PCBs were potent inhibitors of 4-nitrophenol sulfonation but poor substrates in human liver cytosol, and suggested that OH-PCBs may inhibit the sulfation rate of those xenobiotics sulfated by SULT1A1.

The natural anthraquinones from Rheum palmatum induced the metabolic disorder of melatonin by inhibiting human CYP and SULT enzymes.

Melatonin (Mel) as an endogenous hormone, has been widely used in clinic for multiple therapeutic purposes. Further, the natural anthraquinones were widespread in various plants including herbs, foods, and some flavoring agents. The present work aims to evaluate the metabolic disorder of Mel caused by various common herbs and further identify their underlying mechanism. More importantly, the relationships between inhibitory activity and their structures were also investigated. Our results demonstrate that some herbs containing anthraquinone derivatives exhibited strong inhibition on Mel metabolism. Additionally, five anthraquinones from R. palmatum could inhibit phase I and II metabolism of Mel with a mixed inhibition kinetic model based on the mechanism of inhibiting human CYP1A1, 1A2, and SULT1A1. At last, the influence of R. palmatum and its five major components on the Mel metabolism were verified in human primary hepatocytes. In conclusion, our studies elucidated that herbs or foods containing abundant anthraquinones such as R. palmatum will cause a metabolic disorder of Mel, and should be avoided to combined application with Mel in clinic.

Progenitor-derived hepatocyte-like (B-13/H) cells metabolise 1'-hydroxyestragole to a genotoxic species via a SULT2B1-dependent mechanism.

Rat B-13 progenitor cells are readily converted into functional hepatocyte-like B-13/H cells capable of phase I cytochrome P450-dependent activation of pro-carcinogens and induction of DNA damage. The aim of the present study was to investigate whether the cells are also capable of Phase II sulphotransferase (SULT)-dependent activation of a pro-carcinogen to an ultimate carcinogen. To this end we therefore examined the bioactivation of the model hepatic (hepato- and cholangio-) carcinogen estragole and its proximate SULT1A1-activated genotoxic metabolite 1'-hydroxyestragole. Exposing B-13 or B-13/H cells to estragole (at concentrations up to 1mM) resulted in the production of low levels of 1'-hydroxyestragole, but did not result in detectable DNA damage. Exposing B-13/H cells - but not B-13 cells - to 1'-hydroxyestragole resulted in a dose-dependent increase in DNA damage in comet assays, confirmed by detection of N(2)-(trans-isoestragol-3'-yl)-2'-deoxyguanosine adducts. Genotoxicity was inhibited by general SULT inhibitors, supporting a role for SULTS in the activation of 1-hydroxyestragole in B-13/H cells. However, B-13 and B-13/H cells did not express biologically significant levels of SULT1A1 as determined by qRT-PCR, Western blotting and its associated 7-hydroxycoumarin sulphation activity. B-13 and B-13/H cells expressed - relative to intact rat liver - high levels of SULT2B1 (primarily the b isoform) and SULT4A1 mRNAs and proteins. B-13 and B-13/H cells also expressed the 3'-phosphoadenosine 5'-phosphosulphate synthase 1 required for the generation of activated sulphate cofactor 3'-phosphoadenosine 5'-phosphosulphate. However, only B-13/H cells expressed functional SULT activities towards SULT2B1 substrates DHEA, pregnenolone and 4 methylumbelliferone. Since liver progenitor cells are bi-potential and also form cholangiocytes, we therefore hypothesised that B-13 cells express a cholangiocyte-like SULT profile. To test this hypothesis, the expression of SULTs was examined in liver by RT-PCR and immunohistochemistry. SULT2B1 - but not SULT1A1 - was determined to be expressed in both rat and human cholangiocytes. Since 1'-hydroxyestragole exposure readily produced DNA injury in B-13/H cells, these data suggest that cholangiocarcinomas generated in rats fed estragole may be dependent, in part, on SULT2B1 activation of the 1'-hydroxyestragole metabolite.

In vitro inhibition of human hepatic and cDNA-expressed sulfotransferase activity with 3-hydroxybenzo[a]pyrene by polychlorobiphenylols.

Sulfonation is a major phase II biotransformation reaction. In this study, we found that several polychlorobiphenylols (OH-PCBs) inhibited the sulfonation of 3-hydroxybenzo[a]pyrene (3-OH-BaP) by human liver cytosol and some cDNA-expressed sulfotransferases. At concentrations > 0.15 microM, 3-OH-BaP inhibited its own sulfonation in cytosol fractions that were genotyped for SULT1A1 variants, as well as with expressed SULT1A1*1, SULT1A1*2, and SULT1E1, but not with SULT1A3 or SULT1B1. The inhibition fit a two-substrate kinetic model. We examined the effects of OH-PCBs on the sulfonation of 0.1 or 1.0 microM 3-OH-BaP, noninhibitory and inhibitory substrate concentrations, respectively. At the lower 3-OH-BaP concentration, OH-PCBs with a 3-chloro-4-hydroxy substitution pattern were more potent inhibitors of cytosolic sulfotransferase activity [with concentrations that produced 50% inhibition (IC50) between 0.33 and 1.1 microM] than were OH-PCBs with a 3,5-dichloro-4-hydroxy substitution pattern, which had IC50 values from 1.3 to 6.7 microM. We found similar results with expressed SULT1A1*1 and SULT1A1*2. The OH-PCBs were considerably less potent inhibitors when assay tubes contained 1.0 microM 3-OH-BaP. The inhibition mechanism was noncompetitive, and our results suggested that the OH-PCBs competed with 3-OH-BaP at an inhibitory site on the enzyme. The OH-PCBs tested inhibited sulfonation of 3-OH-BaP by SULT1E1, but the order of inhibitory potency was different than for SULT1A1. SULT1E1 inhibitory potency correlated with the dihedral angle of the OH-PCBs. The OH-PCBs tested were generally poor inhibitors of SULT1A3- and SULT1B1-dependent activity with 3-OH-BaP. These findings demonstrate an interaction between potentially toxic hydroxylated metabolites of PCBs and polycyclic aromatic hydrocarbons, which could result in reduced clearance by sulfonation.

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.