Aryl Sulfotransferase Research Articles

Prokaryotic and Eukaryotic Aryl Sulfotransferases: Sulfation of Quercetin and Its Derivatives

AbstractTwo types of sulfotransferases, namely recombinant rat liver aryl sulfotransferase AstIV and bacterial aryl sulfotransferase from Desulfitobacterium hafniense, were used for the sulfation of quercetin, its glycosylated derivatives (isoquercitrin and rutin), and dihydroquercetin ((+)‐taxifolin). The rat liver enzyme was able to sulfate only quercetin and taxifolin, whereas the quercetin glycosides remained intact. The D. hafniense enzyme sulfated isoquercitrin and rutin selectively at the C‐4′ position of the catechol moiety with very good yields. Taxifolin was sulfated at the C‐4′ position and a minor amount of the C‐3′ isomer was formed. Sulfation of quercetin proceeded preferentially at the C‐3′ position, but a lower proportion of the C‐4′ isomer was formed as well. A detailed analysis of the kinetics of this reaction is provided and a full structural analysis of all products is presented.

Constitutive or Inducible Protective Mechanisms against UV-B Radiation in the Brown Alga Fucus vesiculosus? A Study of Gene Expression and Phlorotannin Content Responses.

A role as UV sunscreens has been suggested for phlorotannins, the phenolic compounds that accumulate in brown algae in response to a number of external stimuli and take part in cell wall structure. After exposure of the intertidal brown alga Fucus vesiculosus to artificial UV-B radiation, we examined its physiological responses by following the transcript level of the pksIII gene encoding a phloroglucinol synthase, likely to be involved in the first step of phlorotannins biosynthesis. We also monitored the expression of three targeted genes, encoding a heat shock protein (hsp70), which is involved in global stress responses, an aryl sulfotransferase (ast), which could be involved in the sulfation of phlorotannins, and a vanadium bromoperoxidase (vbpo), which can potentially participate in the scavenging of Reactive Oxygen Species (ROS) and in the cross-linking and condensation of phlorotannins. We investigated whether transcriptional regulation of these genes is correlated with an induction of phlorotannin accumulation by establishing metabolite profiling of purified fractions of low molecular weight phlorotannins. Our findings demonstrated that a high dose of UV-B radiation induced a significant overexpression of hsp70 after 12 and 24 hours following the exposure to the UV-B treatment, compared to control treatment. The physiological performance of algae quantified by the photosynthetic efficiency (Fv/Fm) was slightly reduced. However UV-B treatment did not induce the accumulation of soluble phlorotannins in F. vesiculosus during the kinetics of four weeks, a result that may be related to the lack of induction of the pksIII gene expression. Taken together these results suggest a constitutive accumulation of phlorotannins occurring during the development of F.vesiculosus, rather than inducible processes. Gene expression studies and phlorotannin profiling provide here complementary approaches to global quantifications currently used in studies of phenolic compounds in brown algae.

In vitro Metabolic Modulation of Aryl Sulfotransferases by Pharmaceutical Excipients

Pharmaceutical excipients (PEs) are an essential part of drug products to make them have desirable characteristics. Most pharmaceutical excipients have been considered as biologically inert. However, recent studies showed that PEs affect pharmacokinetic behaviors of drugs in several manners when absorbed through gastrointestinal (GI) tract. For example, oral administration of the excipients such as nonionic surfactants could modulate cytochrome P450 enzymes, in GI tract and liver, and p-glycoprotein (p-gp) leading to changes in pharmacokinetic characteristics in vitro and in vivo. Sulfotransferase (SULT) is one of the phase II metabolism enzymes mainly found in the liver and small intestine and catalyzes the transfer of sulfuryl groups from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to hydroxyl or amino groups of the compounds. The biological functions of SULT include metabolism and detoxification of xenobiotics by sulfation. It can therefore be anticipated that inhibitors or enhancers of SULT alter the metabolic profiles of substrate drugs susceptive to the enzymes which may decrease or increase the bioavailability of the drug. For example, SULT1A1 and SULT1A3 play important roles in the pre-systemic inactivation of β2 agonists in the liver and intestine. Thus, inhibition of SULTs can lead to an increase in the bioavailability of β2 agonists. In this study, we attempted to investigate the possibility of the modulation of SULT by a wide range of PEs frequently used in the formulation of oral drug delivery systems to improve the bioavailability of drugs that are extensively degraded by SULTs.

Identification of interspecific differences in phase II reactions: determination of metabolites in the urine of 16 mammalian species exposed to environmental pyrene.

Interspecific differences in xenobiotic metabolism are a key to determining relative sensitivities of animals to xenobiotics. However, information on domesticated livestock, companion animals, and captive and free-ranging wildlife is incomplete. The present study evaluated interspecific differences in phase II conjugation using pyrene as a nondestructive biomarker of polycyclic aromatic hydrocarbon (PAH) exposure. Polycyclic aromatic hydrocarbons and their metabolites have carcinogenic and endocrine-disrupting effects in humans and wildlife and can have serious consequences. The authors collected urine from 16 mammalian species and analyzed pyrene metabolites. Interspecific differences in urinary pyrene metabolites, especially in the concentration and composition of phase II conjugated metabolites, were apparent. Glucuronide conjugates are dominant metabolites in the urine of many species, including deer, cattle, pigs, horses, and humans. However, they could not be detected in ferret urine even though the gene for ferret Uridine 5'-diphospho-glucuronosyltransferase (UDP-glucuronosyltransferase, UGT) 1A6 is not a pseudogene. Sulfate conjugates were detected mainly in the urine of cats, ferrets, and rabbits. Interestingly, sulfate conjugates were detected in pig urine. Although pigs are known to have limited aryl sulfotransferase activity, the present study demonstrated that pig liver was active in 1-hydroxypyrene sulfation. The findings have some application for biomonitoring environmental pollution.

Comparison of dopamine with levodopa in the induction of aryl sulfotransferases and estrogen sulfotransferase in rat liver

Comparison of dopamine with levodopa in the induction of aryl sulfotransferases and estrogen sulfotransferase in rat liver

Aryl Sulfotransferase from Haliangium ochraceum: A Versatile Tool for the Sulfation of Small Molecules

AbstractSulfation is an important molecular modification that regulates essential cellular processes and is also implicated in numerous pathological processes. The enzymes responsible for this reaction in living organisms are sulfotransferases. The gene Hoch_5094 from Haliangium ochraceum is annotated as a putative sulfotransferase. The arylsulfotransferase codified by this gene (HocAST) was expressed heterologously in E. coli and showed aryl sulfotransferase activity. Circular dichroism analysis of HocAST showed a main α/β secondary structure that agrees with the overall structure of other cytosolic sulfotransferases. Interestingly, HocAST was able to use both p‐nitrophenyl sulfate and 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) as sulfuryl donors contrary to that of aryl sulfate sulfotransferase, which cannot use PAPS as a donor. Regarding the specificity towards the acceptor, HocAST has shown quite a wide scope and was able to accept several mono‐ and dihydroxylated phenols and other phosphorylated compounds as substrates.

Structural and Mechanistic Insights into the PAPS-Independent Sulfotransfer Catalyzed by Bacterial Aryl Sulfotransferase and the Role of the DsbL/DsbI System in Its Folding

Bacterial aryl sulfotransferases (ASSTs) catalyze sulfotransfer from a phenolic sulfate to a phenol. These enzymes are frequently found in pathogens and upregulated during infection. Their mechanistic understanding is very limited, and their natural substrates are unknown. Here, the crystal structures of Escherichia coli CFT073 ASST trapped in its presulfurylation state with model donor substrates bound in the active site are reported, which reveal the molecular interactions governing substrate recognition. Furthermore, spectroscopic titrations with donor substrates and sulfurylation kinetics of ASST illustrate that this enzyme binds substrates in a 1:1 stoichiometry and that the active sites of the ASST homooligomer act independently. Mass spectrometry and crystallographic experiments of ASST incubated with human urine demonstrate that urine contains a sulfuryl donor substrate. In addition, we examined the capability of the two paralogous dithiol oxidases present in uropathogenic E. coli CFT073, DsbA, and the ASST-specific enzyme DsbL, to introduce the single, conserved disulfide bond into ASST. We show that DsbA and DsbL introduce the disulfide bond into unfolded ASST at similar rates. Hence, a chaperone effect of DsbL, not present in DsbA, appears to be responsible for the dependence of efficient ASST folding on DsbL in vivo. The conservation of paralogous dithiol oxidases with different substrate specificities in certain bacterial strains may therefore be a consequence of the complex folding pathways of their substrate proteins.

Using structure- and Ligand-based pharmacophores as filters to discriminate Human Aryl Sulfotransferase 1A1 (SUL1A1) binders into substrates and inhibitors

Predicting metabolictransformation is one of major challenges in drug discovery [1]. Sulfotransferase 1A1 (SULT1A1), one of phase II metabolismenzymes, is the major SULT in adult liver catalysis [2]. It metabolizes many endogenous compounds and is relevant in carcinogenesis due to its ability to modify diverse promutagen and procarcinogen xenobiotics [3]. In order to make a discriminative model that classifies group of SULT1A1 binders into substrates and inhibitors, a combination of structure, ligand-based, and docking based pharmacophores have been generated and validated by Ligandscout [4]. On one hand, structure-based pharmacophores have been derived from PDB files of good binders (substrates and inhibitors). On the other hand, ligand-based interaction maps have been conducted from some drug classes that show different substrate/inhibitor activity towards SULT1A1.Finally, highly active substrates and inhibitors have been docked into the enzyme using GOLD [5], and subsequently molecular interaction fields have been developed for the most plausible poses. As a retrospective validation, all pharmacophores simultaneously have been used to screen more than 100 SULT1A1 binders covering several activity classes and different chemical scaffolds. The model showed good discriminative power to differentiate between inhibitors, substrates and mixed substrates/inhibitors.

Enzymatic preparation of silybin phase II metabolites: sulfation using aryl sulfotransferase from rat liver

Aryl sulfotransferase IV (AstIV) from rat liver was overexpressed in Escherichia coli and purified to homogeneity. Using the produced mammalian liver enzyme, sulfation-the Phase II conjugation reaction-of optically pure silybin diastereoisomers (silybin A and B) was tested. As a result, silybin B was sulfated yielding 20-O-silybin B sulfate, whereas silybin A was completely resistant to the sulfation reaction. Milligram-scale sulfation of silybin B was optimized employing resting E. coli cells producing AstIV, thus avoiding the use of expensive 3'-phosphoadenosine-5'-phosphate cofactor and laborious enzyme purification. Using this approach, we were able to reach 48 % conversion of silybin B into its 20-sulfate within 24 h. The sulfated product was isolated by solid phase extraction and its structure was characterized by HRMS and NMR. Sulfation reaction of silybin appeared strictly stereoselective; only silybin B was sulfated by AstIV.

MP2//DFT calculations of interaction energies between acetaminophen and acetaminophen analogues and the aryl sulfotransferase active site

Paracetamol, or acetaminophen, is a commonly used analgesic and antipyretic. Cresols such as p-cresol, o-cresol, and m-cresol can compete with acetaminophen for metabolism via several pathways, including via sulfation by aryl sulfotransferase. We have applied the MP2 method to study the interaction of acetaminophen and these cresol analogues with the active site of aryl sulfotransferase. Docking and BHandHLYP/6-31G optimization were used to find the structures of the ligand–protein complexes assuming a static active site. Interaction energies between the ligands and each of the amino acids in the active site were calculated using MP2 with a basis set of 6-311+g*. Further optimizations were then performed to allow flexibility of the amino acid residue side-chains in the active site and interaction energies were calculated for these complexes as well. p-Cresol is shown to compete with acetaminophen for this metabolic pathway, in agreement with experiment. We also show that m-cresol should be competitive for this metabolic pathway.

Caffeine induction of sulfotransferases in rat liver and intestine

Sulfotransferases (SULTs) are important phase II drug-metabolizing enzymes. Regulation of SULTs by hormones and other endogenous molecules is relatively well understood, while xenobiotic induction of SULTs is not well studied. Caffeine is one of the most widely consumed psychoactive substances. However, SULT regulation by caffeine has not been reported. In this report, male and female rats were treated with different oral doses of caffeine (2, 10, 50 mg kg⁻¹ per day) for 7 days. Western blot and real-time RT-PCR were used to investigate the changes in SULT protein and mRNA expression following the caffeine treatment. Caffeine induced both rat aryl sulfotransferase (rSULT1A1, AST-IV) and rat hydroxysteroid sulfotransferase (rSULT2A1, STa) in the liver and intestine of female rats in a dose-dependent manner. Caffeine induction of rSULT1A1 and rSULT2A1 in the female rat intestine was much stronger than that in the liver. Although caffeine induced rSULT1A1 significantly in the male rat liver, it did not significantly induce rSULT2A1. In male rat intestine, caffeine significantly induced rSULT2A1. The different SULTs induction patterns in male and female rats suggest that the regulation of rat SULTs by caffeine may be affected by different hormone secretion patterns and levels. Our results suggest that consumption of caffeine can induce drug metabolizing SULTs in drug detoxification tissues.

Proteomic analysis of chronic restraint stress-induced Gan (肝)-stagnancy syndrome in rats

To analyze the proteomic characteristics of Gan (肝)-stagnancy syndrome (GSS) by seeking the differential protein in blood and tissues of GSS model rats. GSS model rats were established by chronic restraint stress, keeping rats in restrain chamber for 6 h every day for 21 successive days. Their blood and liver samples were collected at the end of experiment for differential protein detection with methods of isoelectrofocusing and polyacrylamide SDS-PAGE, silver staining, and scanning. The gel images were analyzed with Imagemaster 2D Elite software, and the excavated differential protein spots were identified with matrix assistant laser resolving TOF mass spectrometry, Western blot, ELISA, and RT-PCR, respectively. A method for isolating the protein in blood serum and tissues by two-dimensional gel electrophoresis was established and optimized. Six serum proteins and three liver proteins that differentially expressed were identified. The down-regulated differential proteins in serum of GSS model rats were serum albumin precursor, beta 1 globin, antibody against muscle acetylcholine receptor, Ig lambda-2 C region, and transthyretin (TTR), and those in liver tissue were aryl sulfotransferase, enoyl-CoA hydratase, and TTR. TTR down-regulation was found in both serum and liver. Preliminary biological information analysis showed that these differential proteins involved in immune, neuroendocrine, nutrition, and substance metabolism. Proteomic analysis of differential proteins showed that TTR, aryl sulfotransferase, and enoyl-CoA hydratase expressions are downregulated in the GSS model rats, suggesting that the susceptibility of cancer could be enhanced by chronic stress.

The PAPS-Independent Aryl Sulfotransferase and the Alternative Disulfide Bond Formation System in Pathogenic Bacteria

Sulfurylation of biomolecules (often termed sulfonation or sulfation) has been described in many organisms in all kingdoms of life. To date, most studies on sulfotransferases, the enzymes catalyzing sulfurylation, have focused on 3'-phosphate-5'-phosphosulfate (PAPS)-dependent enzymes, which transfer the sulfuryl group from this activated anhydride to hydroxyl groups of acceptor molecules. By contrast, the PAPS-independent aryl sulfotransferases (ASSTs) from bacteria, which catalyze sulfotransfer from phenolic sulfate esters to another phenol in the bacterial periplasm, were not well characterized until recently, although they were first described in 1986 in a search for nonhepatic sulfurylation processes. Recent studies revealed that this unusual class of sulfotransferases differs profoundly in both molecular structure and catalytic mechanism from PAPS-dependent sulfotransferases, and that ASSTs from certain bacterial pathogens are upregulated during infection. In this review, we summarize the literature on the roles of sulfurylation in prokaryotes and analyze the occurrence of ASSTs and their dependence on Dsb proteins catalyzing oxidative folding in the periplasm. Furthermore, we discuss structural differences and similarities between aryl sulfotransferases and PAPS-dependent sulfotransferases.

A structural and biochemical basis for PAPS-independent sulfuryl transfer by aryl sulfotransferase from uropathogenic Escherichia coli

Sulfotransferases are a versatile class of enzymes involved in numerous physiological processes. In mammals, adenosine 3'-phosphate-5'-phosphosulfate (PAPS) is the universal sulfuryl donor, and PAPS-dependent sulfurylation of small molecules, including hormones, sugars, and antibiotics, is a critical step in hepatic detoxification and extracellular signaling. In contrast, little is known about sulfotransferases in bacteria, which make use of sulfurylated molecules as mediators of cell-cell interactions and host-pathogen interactions. Bacterial arylsulfate sulfotransferases (also termed aryl sulfotransferases), in contrast to PAPS-dependent sulfotransferases, transfer sulfuryl groups exclusively among phenolic compounds in a PAPS-independent manner. Here, we report the crystal structure of the virulence factor arylsulfate sulfotransferase (ASST) from the prototypic, pyelonephritogenic Escherichia coli strain CFT073 at 2.0-A resolution, and 2 catalytic intermediates, at 2.1-A and 2.4-A resolution, with substrates bound in the active site. ASST is one of the largest periplasmic enzymes and its 3D structure differs fundamentally from all other structurally characterized sulfotransferases. Each 63.8-kDa subunit of the ASST homodimer comprises a 6-bladed beta-propeller domain and a C-terminal beta-sandwich domain. The active sites of the dimer are situated at the center of the channel formed by each beta-propeller and are defined by the side chains of His-252, His-356, Arg-374, and His-436. We show that ASST follows a ping-pong bi-bi reaction mechanism, in which the catalytic residue His-436 undergoes transient sulfurylation, a previously unreported covalent protein modification. The data provide a framework for understanding PAPS-independent sulfotransfer and a basis for drug design targeting this bacterial virulence factor.

Effect of Folic Acid on Methotrexate Induction of Sulfotransferases in Rats

Our earlier investigation showed that MTX is an inducer of rat and human sulfotransferases. Here we report that folic acid treatment inhibited MTX induction of aryl sulfotransferase (AST-IV) in female rat liver and hydroxysteroid sulfotransferase (STa) in male rat liver. This is important for understanding the clinical mechanisms of MTX.

Pentachlorophenol inhibits micronuclei induction by 2-acetylaminofluorene but not by thioacetamide

Our study examined the capacity of pentachlorophenol (PCP) to inhibit the ability of 2-acetylaminofluorene (2-AAF) and thioacetamide (TAA) to induce micronuclei in mouse bone marrow cells in vivo. 2-AAF (5.6 mg/kg) and TAA (60 mg/kg) were administered intra-peritoneally (i.p.) to Mus musculus males (BALB/c), and the frequencies of polychromatic erythrocytes with micronuclei (PCE-MN) 24 h after injection were analyzed. Treatment with 2-AAF or TAA resulted in high PCE-MN frequencies in comparison with untreated and negative controls (19.9 and 21.6‰, respectively, versus ≈3‰). Pretreatment with a single PCP dose (44 mg/kg) 24 h prior to the 2-AAF administration virtually eliminated micronuclei formation by 2-AAF, although it had no inhibitory effect on TAA-induced micronuclei. Animals receiving cyclophosphamide (CP) served as positive control. Since PCP is known to inhibit arylsulfotransferase (AST) activity, which is involved in 2-AAF activation, this mechanism most likely produced the results with PCP and 2-AAF. Our results also are consistent with a different pathway involved in TAA induction of micronuclei, one that is not inhibited by PCP.

In vivo and in vitro oxidative regulation of rat aryl sulfotransferase IV (AST IV)

Sulfotransferase catalyzed sulfation is important in the regulation of different hormones and the metabolism of hydroxyl containing xenobiotics. In the present investigation, we examined the effects of hyperoxia on aryl sulfotransferase IV in rat lungs in vivo. The enzyme activity of aryl sulfotransferase IV increased 3- to 8-fold in >95% O2 treated rat lungs. However, hyperoxic exposure did not change the mRNA and protein levels of aryl sulfotransferase IV in lungs as revealed by Western blot and RT-PCR. This suggests that oxidative regulation occurs at the level of protein modification. The increase of nonprotein soluble thiol and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios in treated lung cytosols correlated well with the aryl sulfotransferase IV activity increase. In vitro, rat liver cytosol 2-naphthol sulfation activity was activated by GSH and inactivated by GSSG. Our results suggest that Cys residue chemical modification is responsible for the in vivo and in vitro oxidative regulation. The molecular modeling structure of aryl sulfotransferase IV supports this conclusion. Our gel filtration chromatography results demonstrated that neither GSH nor GSSG treatment changed the existing aryl sulfotransferase IV dimer status in cytosol, suggesting that oxidative regulation of aryl sulfotransferase IV is not caused by dimer-monomer status change.

All‐trans Retinoic Acid Induction of Sulfotransferases

All-trans retinoic acid is the bioactive form of vitamin A (retinol). Retinoids have been used clinically as therapeutic agents against a number of cancers. Retinoids have been reported to induce the phase I drug metabolizing enzymes, cytochrome P-450s. In contrast, effects of retinoids on sulfotransferases have not been as well studied. The present investigation evaluates the role of retinoic acid on the expression of aryl sulfotransferase IV and hydroxysteroid sulfotransferase a in male and female Sprague-Dawley rat liver and intestine. Cultured human hepatic carcinoma cells (Hep G2) and intestinal carcinoma cells (Caco-2) were also used to study retinoic acid's effect on simple phenol sulfating sulfotransferase, dehydroepiandrosterone sulfotransferase and oestrogen sulfotransferase. Enzyme assay and Western blot were used to determine sulfotransferase protein expression. Retinoic acid induced aryl sulfotransferase IV in liver of female rats and sulfotransferase a in liver of male rats. Intestinal rat aryl sulfotransferase IV and sulfotransferase a in male rats and intestinal aryl sulfotransferase IV in female rats were also induced after retinoic acid treatment. In Hep G2 and Caco-2 cells, retinoic acid differentially induced the three human sulfotransferase isoforms. In general, intestinal sulfotransferases were found to be more responsive than hepatic sulfotransferases to retinoic acid treatment. mRNA expressions were investigated using reverse transcription polymerase chain reaction with gene specific primers. Reverse transcription polymerase chain reaction results are in good agreement with enzyme activity and Western blot results. This suggests that retinoic acid induction of sulfotransferases is at the transcriptional level.

Influence of phenylalanines 77 and 138 on the stereospecificity of aryl sulfotransferase IV.

Aryl sulfotransferase (AST) IV (also named tyrosine-ester sulfotransferase and ST1A1) is a major phenol sulfotransferase in the rat, and it catalyzes the sulfation of many drugs, carcinogens, and other xenobiotics that contain phenol, benzylic alcohol, N-hydroxy arylamine, and oxime functional groups. Previous work discovered a stereospecificity of AST IV toward the enantiomers of 1,2,3,4-tetrahydro-1-naphthol and varying degrees of stereoselectivity with other chiral benzylic alcohols. The studies described here were directed toward understanding the roles of specific amino acid residues at the substrate binding site in determining the stereoselectivity of this sulfotransferase isoform. Docking experiments with a homology model of AST IV revealed three amino acid residues, Phe77, Phe138, and Tyr236, that may potentially be important for interactions with substituents on the chiral carbon of a benzylic alcohol serving as a sulfuryl acceptor, thereby imparting stereoselectivity. To test this hypothesis, mutants were constructed wherein each of the above residues was substituted with alanine. Kinetic studies on the sulfation of the enantiomers of 1,2,3,4-tetrahydro-1-naphthol indicated that the stereospecificity of the sulfotransferase was altered by the substitutions of alanine for either Phe77 or Phe138, but stereospecificity was maintained by alanine substitution at Tyr236. Molecular models of the mutant enzymes interacting with enantiomers of 1,2,3,4-tetrahydro-1-naphthols and with 2-naphthol indicate that Phe77 and Phe138 provide significant steric interactions at the active site that both enhance catalytic efficiency and impart stereospecificity in molecular recognition of substrates and inhibitors.

Transactivation of glucocorticoid-inducible rat aryl sulfotransferase (SULT1A1) gene transcription.

The purpose of the current study was to establish the role of the glucocorticoid receptor (GR) and androgen receptor (AR) transcription factors in the transactivation of rat aryl sulfotransferase (SULT1A1) gene transcription and to identify the functional hormone-responsive element(s) in the SULT1A1 gene. A cis-acting inverted repeat with three intervening bases (IR3) was identified in the 5'-flanking of the SULT1A1 gene that mediates the transactivation of SULT1A1 gene transcription by both the GR and AR. CV-1 cells were cotransfected with SULT1A1-luciferase reporter plasmids and either wild-type or mutant GR or AR expression constructs. In cotransfectants expressing the wild-type GR, treatment with triamcinolone acetonide produced an approximately 4- to 6-fold induction of luciferase activity in IR3-containing SULT1A1 reporter plasmids. IR3-containing SULT1A1 reporter constructs were also activated by treatment with the synthetic androgen R1881 in cells cotransfected with wild-type but not mutant AR. In primary cultured rat hepatocytes, androgen-inducible expression of IR3-containing SULT1A1 reporter plasmids required cotransfection with AR expression plasmid. Targeted disruption of the SULT1A1 IR3 by mutation of a conserved GT sequence in the 3' half-site of the element ablated GR and AR responsiveness. These results indicate that a proximal IR3 element in the 5'-flanking region of the rat SULT1A1 gene is sufficient for the transactivation of SULT1A1 gene transcription by the GR and AR, and that relative to the GR, functional AR activity is reduced in primary cultured rat hepatocytes.