Expression of functional sulfotransferases (SULT) 1A1, 1A3, 1B1, 1C2, 1E1, and 2A1 in common marmosets.

Cytosolic sulfotransferases (SULTs), which mediate the conjugation of drugs with 3'-phosphoadenosine-5'-phosphosulfate, have been characterized in humans and cynomolgus monkeys. However, SULTs remain to be evaluated in common marmosets, a species of non-human primate often employed in drug metabolism and pharmacokinetic studies of endogenous and exogenous compounds. In this study, marmoset SULT1A1, 1A3, 1B1, 1C2, 1E1, and 2A1 cDNAs were isolated and characterized, based on genome data. The deduced amino acid sequences of these marmoset SULT cDNAs had high identities (90-95%) with their human orthologs, except for marmoset SULT2A1, which was only 81% identical to human SULT2A1. The amino acid sequences of the orthologs of these six SULTs in marmosets, monkeys, and humans were closely clustered in a phylogenetic tree. The structures and genomic organizations of marmoset SULT genes were similar to those of their human orthologs. Among the five marmoset tissues analyzed, SULT mRNAs showed typical expression patterns. The most abundant SULT mRNAs were SULT1B1 in liver, small intestine, and kidney; SULT1E1 in lung; and SULT1A3 in brain. Recombinant marmoset SULT1A1, 1A3, 1B1, 1C2, 1E1, and 2A1 proteins expressed in bacterial cytosolic fractions mediated sulfate conjugations with 3'-phosphoadenosine-5'-phosphosulfate of the following typical human SULT substrates: dopamine, 1-naphthol, p-nitrophenol, estradiol, and dehydroepiandrosterone. Taken together, these wide-ranging results suggest functional and molecular similarities of SULTs among marmosets, monkeys, and humans.

Expression of clinically relevant drug-metabolizing enzymes along the human intestine and their correlation to drug transporters and nuclear receptors: An intra-subject analysis.

The oral bioavailability of many drugs is highly influenced not only by hepatic but also by intestinal biotransformation. To estimate the impact of intestinal phase I and II metabolism on oral drug absorption, knowledge on the expression levels of the respective enzymes is an essential prerequisite. In addition, the potential interplay of metabolism and transport contributes to drug disposition. Both mechanisms may be subjected to coordinative regulation by nuclear receptors, leading to unwanted drug-drug interactions due to induction of intestinal metabolism and transport. Thus, it was the aim of this study to comprehensively analyse the regional expression of clinically relevant phase I and II enzymes along the entire human intestine and to correlate these data to expression data of drug transporters and nuclear receptors of pharmacokinetic relevance. Gene expression of 11 drug-metabolizing enzymes (CYP2B6, 2C8, 2C9, 2C19, 2D6, 3A4, 3A5, SULT1A, UGT1A, UGT2B7, UGT2B15) was studied in duodenum, jejunum, ileum and colon from six organ donors by real-time RT-PCR. Enzyme expression was correlated with expression data of the nuclear receptors PXR, CAR and FXR as well as drug transporters observed in the same cohort. Intestinal expression of all studied metabolizing enzymes was significantly higher in the small intestine compared to colonic tissue. CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4/5, SULT1A, UGT1A and UGT2B7 expression increased from the duodenum to jejunum but was markedly lower in the ileum. In the small intestine, that is, the predominant site of drug absorption, the highest expression has been observed for CYP3A4, CYP2C9, SULT1A and UGT1A. In addition, significant correlations were found between several enzymes and PXR as well as ABC transporters in the small intestine. In conclusion, the observed substantial site-dependent intestinal expression of several enzymes may explain regional differences in intestinal drug absorption. The detected correlations between intestinal enzymes, transporters and nuclear receptors provide indirect evidence for their coordinative expression, regulation and function in the human small intestine.

Dopamine-induced SULT1A3/4 promotes EMT and cancer stemness in hepatocellular carcinoma.

Hepatocellular carcinoma has the second highest incidence rate among malignant cancers in China. Hepatocellular carcinoma development is complex because of the metabolism disequilibrium involving SULT1A3/4, a predominant sulfotransferase that metabolizes sulfonic xenobiotics and endogenous catecholamines. However, the correlation between SULT1A3/4 and hepatocellular carcinoma progression is unclear. By utilizing immunofluorescence and immunohistochemical analysis, we found that in nine hepatocellular carcinoma clinical specimens, SULT1A3/4 was abundantly expressed in tumor tissues compared to that in the adjacent tissues. Moreover, liver cancer cells (HepG2, MHCC97-L, and MHCC97-H) had higher basal expression of SULT1A3/4 than immortalized liver cells (L02 and Chang liver). Ultra-high-pressure liquid chromatography-tandem mass spectrometry assay results further revealed that the concentration of dopamine (a substrate of SULT1A3/4) was negatively correlated with SULT1A3/4 protein expression. As a transcriptional regulator of SULT1A3/4 in turn, dopamine was used to induce SULT1A3/4 in vitro. Interestingly, dopamine significantly induced SULT1A3/4 expression in liver cancer HepG2 cells, while decreased that in L02 cells. More importantly, the expression levels of epithelial-mesenchymal transition biomarkers (N-cadherin and vimentin) and cell stemness biomarkers (nanog, sox2, and oct3/4) considerably increased in HepG2 with dopamine-induced SULT1A3/4, whereas in L02, epithelial-mesenchymal transition and cancer stem cell-associated proteins were contrarily decreased. Furthermore, invasion and migration assays further revealed that dopamine-induced SULT1A3/4 dramatically stimulated the metastatic capacity of HepG2 cells. Our results implied that SULT1A3/4 exhibited bidirectional effect on tumor and normal hepatocytes and may thus provide a novel strategy for hepatocellular carcinoma clinical targeting. In addition, SULT1A3/4 re-expression could serve as a biomarker for hepatocellular carcinoma prognosis.

Differential sensitivities of bladder cancer cell lines to resveratol are unrelated to its metabolic profile.

Resveratrol (RV) is a natural polyphenol compound with a wide range of activities, including inhibition of human bladder cancer (HBC) cell growth. Because RV is rapidly metabolized and has poor bioavailability, it is unclear whether the antitumor activity is due to RV or its metabolites. We therefore used liquid chromatography-mass spectroscopy, qRT-PCR, immunocytochemistry and western blotting to evaluate the metabolic profile and biotransformation of RV in the T24 and EJ HBC cell lines. Both T24 and EJ cells generated the same RV metabolite, RV monosulfate (RVS), and both exhibited upregulation of the RV-associated metabolic enzyme SULT1A1 (sulfotransferase). Despite these similarities, T24 cells were more sensitive to RV than EJ cells, yet T24 cells exhibited no sensitivity to an RVS mixture (84.13% RVS). Primary rat bladder epithelial cells showed no adverse effects when exposed to a therapeutic dose (100 µM) of RV. The differences in RV sensitivity between the two HBC cell lines did not reflect differences in the RV metabolic profile or SULT1A1 expression. Because RV exhibited stronger antitumor activity and better safety than RVS, we conclude that RV has significant therapeutic potential for HBC treatment, provided individual differences are considered during clinical research and application.

Sulfonation of raloxifene in HEK293 cells overexpressing SULT1A3: Involvement of breast cancer resistance protein (BCRP/ABCG2) and multidrug resistance-associated protein 4 (MRP4/ABCC4) in excretion of sulfate metabolites.

Excretion of sulfate metabolites is an essential process in disposition of raloxifene via the sulfonation pathway. However, the transporters responsible for excretion of raloxifene sulfates remain undefined. Here, sulfonation of raloxifene and excretion of its sulfate metabolites were investigated using SULT1A3-overexpressing HEK293 cells (or SULT293 cells) with significant expression of BCRP and MRP4. SULT293 cell lysate catalyzed the sulfonation of raloxifene at both 6-OH and 4'-OH groups, generating raloxifene-6-sulfate (R-6-S) and raloxifene-4'-sulfate (R-4'-S), respectively. Sulfate formation followed the Michaelis-Menten kinetics (Km = 0.49 µM and Vmax = 5.79 pmol/min/mg for R-6-S; Km = 0.33 µM and Vmax = 1.25 pmol/min/mg for R-4'-S). As expected, the recombinant SULT1A3 enzyme showed a high similarity in raloxifene sulfonation profiles with the lysate preparation. Ko143, a selective inhibitor of BCRP, significantly decreased the excretion rates of raloxifene sulfates (maximal 66.1%) while increasing the intracellular sulfates (maximal 282%). As a result, the apparent efflux clearance (CLef,app, representing the efflux efficiency of raloxifene sulfates) was substantially reduced (maximal 85.6%). Likewise, the pan-MRP inhibitor MK-571 significantly deceased the excretion rates (maximal 69.6%) and CLef,app values (maximal 96.0%) of raloxifene sulfates while increasing the intracellular sulfates (maximal 667%). Further, the short-hairpin RNA (shRNA) targeting BCRP significantly reduced (maximal 35.0%) sulfate excretion. Use of BCRP shRNA also caused significant decreases (maximal 52.5%) in the CLef,app values. Silencing of MRP4 by shRNA led to a substantial alteration in sulfate disposition (i.e., 28.6-37.8% reductions in sulfate excretion, 30.5-59.3% elevations in intracellular sulfates, and 44.8-47.7% deceases in CLef,app values). In conclusion, two sulfate metabolites R-6-S and R-4'-S were generated from raloxifene in SULT293 cells. Cellular excretion of the raloxifene sulfates was mainly mediated by BCRP and MRP4.

The proteome of Hypobaric Induced Hypoxic Lung: Insights from Temporal Proteomic Profiling for Biomarker Discovery.

Exposure to high altitude induces physiological responses due to hypoxia. Lungs being at the first level to face the alterations in oxygen levels are critical to counter and balance these changes. Studies have been done analysing pulmonary proteome alterations in response to exposure to hypobaric hypoxia. However, such studies have reported the alterations at specific time points and do not reflect the gradual proteomic changes. These studies also identify the various biochemical pathways and responses induced after immediate exposure and the resolution of these effects in challenge to hypobaric hypoxia. In the present study, using 2-DE/MS approach, we attempt to resolve these shortcomings by analysing the proteome alterations in lungs in response to different durations of exposure to hypobaric hypoxia. Our study thus highlights the gradual and dynamic changes in pulmonary proteome following hypobaric hypoxia. For the first time, we also report the possible consideration of SULT1A1, as a biomarker for the diagnosis of high altitude pulmonary edema (HAPE). Higher SULT1A1 levels were observed in rats as well as in humans exposed to high altitude, when compared to sea-level controls. This study can thus form the basis for identifying biomarkers for diagnostic and prognostic purposes in responses to hypobaric hypoxia.

Expression of sulfotransferase SULT1A1 in cancer cells predicts susceptibility to the novel anticancer agent NSC-743380.

The small molecule anticancer agent NSC-743380 modulates functions of multiple cancer-related pathways and is highly active in a subset of cancer cell lines in the NCI-60 cell line panel. It also has promising in vivo anticancer activity. However, the mechanisms underlying NSC-743380's selective anticancer activity remain uncharacterized. To determine biomarkers that may be used to identify responders to this novel anticancer agent, we performed correlation analysis on NSC-743380's anticancer activity and the gene expression levels in NCI-60 cell lines and characterized the functions of the top associated genes in NSC-743380-mediated anticancer activity. We found sulfotransferase SULT1A1 is causally associated with NSC-743380's anticancer activity. SULT1A1 was expressed in NSC-743380-sensitive cell lines but was undetectable in resistant cancer cells. Ectopic expression of SULT1A1 in NSC743380 resistant cancer cells dramatically sensitized the resistant cells to NSC-743380. Knockdown of the SULT1A1 in the NSC-743380 sensitive cancer cell line rendered it resistance to NSC-743380. The SULT1A1 protein levels in cell lysates from 18 leukemia cell lines reliably predicted the susceptibility of the cell lines to NSC-743380. Thus, expression of SULT1A1 in cancer cells is required for NSC-743380's anticancer activity and can be used as a biomarker for identification of NSC-743380 responders.

Methamphetamine regulation of sulfotransferase 1A1 and 2A1 expression in rat brain sections.

Sulfotransferase catalyzed sulfation regulates the biological activities of various neurotransmitters/hormones and detoxifies xenobiotics. Rat sulfotransferase rSULT1A1 catalyzes the sulfation of neurotransmitters and xenobiotic phenolic compounds. rSULT2A1 catalyzes the sulfation of hydroxysteroids and xenobiotic alcoholic compounds. In this work, Western blot and real-time RT-PCR were used to investigate the effect of methamphetamine on rSULT1A1 and rSULT2A1 protein and mRNA expression in rat cerebellum, frontal cortex, hippocampus, and striatum. After 1-day treatment, significant induction of rSULT1A1 was observed only in the cerebellum; rSULT2A1 was induced significantly in the cerebellum, frontal cortex, and hippocampus. After 7 days of exposure, rSULT1A1 was induced in the cerebellum, frontal cortex, and hippocampus, while rSULT2A1 was induced significantly in all four regions. Western blot results agreed with the real-time RT-PCR results, suggesting that the induction occurred at the gene transcriptional level. Results indicate that rSULT1A1 and rSULT2A1 are expressed in rat frontal cortex, cerebellum, striatum, and hippocampus. rSULT1A1 and rSULT2A1are inducible by methamphetamine in rat brain sections in a time dependable manner. rSULT2A1 is more inducible than rSULT1A1 by methamphetamine in rat brain sections. Induction activity of methamphetamine is in the order of cerebellum>frontal cortex, hippocampus>striatum. These results suggest that the physiological functions of rSULT1A1 and rSULT2A1 in different brain regions can be affected by methamphetamine.

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.

A new arylsulfate sulfotransferase involved in liponucleoside antibiotic biosynthesis in streptomycetes.

Sulfotransferases are involved in a variety of physiological processes and typically use 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfate donor substrate. In contrast, microbial arylsulfate sulfotransferases (ASSTs) are PAPS-independent and utilize arylsulfates as sulfate donors. Yet, their genuine acceptor substrates are unknown. In this study we demonstrate that Cpz4 from Streptomyces sp. MK730-62F2 is an ASST-type sulfotransferase responsible for the formation of sulfated liponucleoside antibiotics. Gene deletion mutants showed that cpz4 is required for the production of sulfated caprazamycin derivatives. Cloning, overproduction, and purification of Cpz4 resulted in a 58-kDa soluble protein. The enzyme catalyzed the transfer of a sulfate group from p-nitrophenol sulfate (K(m) 48.1 microM, k(cat) 0.14 s(-1)) and methyl umbelliferone sulfate (K(m) 34.5 microM, k(cat) 0.15 s(-1)) onto phenol (K(m) 25.9 and 29.7 mM, respectively). The Cpz4 reaction proceeds by a ping pong bi-bi mechanism. Several structural analogs of intermediates of the caprazamycin biosynthetic pathway were synthesized and tested as substrates of Cpz4. Des-N-methyl-acyl-caprazol was converted with highest efficiency 100 times faster than phenol. The fatty acyl side chain and the uridyl moiety seem to be important for substrate recognition by Cpz4. Liponucleosides, partially purified from various mutant strains, were readily sulfated by Cpz4 using p-nitrophenol sulfate. No product formation could be observed with PAPS as the donor substrate. Sequence homology of Cpz4 to the previously examined ASSTs is low. However, numerous orthologs are encoded in microbial genomes and represent interesting subjects for future investigations.

Expression and functional activities of selected sulfotransferase isoforms in BeWo cells and primary cytotrophoblast cells.

Several cytosolic sulfotransferase enzyme isoforms are functional in placenta but there is limited information available on the utility of cultured trophoblast cells for studying sulfation. The trophoblast cell layer constitutes the rate-determining barrier for trans-placental transfer. The objective of this work was to examine the mRNA expression and enzyme activities of four sulfotransferase isoforms reported to be functional in human placenta (SULT1A1, SULT1A3, SULT1E1, and SULT2A1) in primary cytotrophoblast cells and the trophoblast-like BeWo cell line. Reverse transcription polymerase chain reaction (RT-PCR) was performed to determine mRNA expression. Enzyme activities were assessed using the following substrates: 4-nitrophenol for SULT1A1, dopamine for SULT1A3, 17beta-estradiol for SULT1E1, and dehydroepiandrosterone for SULT2A1. For 4-nitrophenol and dopamine sulfation, apparent K(m) values, response to inhibitors (2,6-dichloro-4-nitrophenol and sodium chloride), and thermal stability profiles indicated that 4-nitrophenol and dopamine sulfation in BeWo cells were being mediated by SULT1A1 and SULT1A3, respectively. SULT1A1 and SULT1A3 were also functional in the cytotrophoblast cells. Both at the protein and at the mRNA levels, SULT1A1 was more abundant in BeWo cells in comparison to the primary cytotrophoblast cells. SULT1E1 and SULT2A1 mRNA were not detected in the cytotrophoblasts. SULT1E1 mRNA was weakly expressed in BeWo but there was negligible functional activity. Although SULT2A1 mRNA was abundantly expressed in BeWo, Western blot and enzyme activities revealed that the protein is not expressed in BeWo cells. The results suggest that the BeWo cells and the cytotrophoblast cells can be used to examine the roles of SULT1A1 and SULT1A3 in placental metabolism.

Trans-resveratrol-mediated inhibition of beta-oestradiol conjugation in MCF-7 cells stably expressing human sulfotransferases SULT1A1 or SULT1E1, and human liver microsomes.

High concentrations of endogenous oestradiol (E2) correlate with the proliferation of cancer cells. Resveratrol (a dietary chemopreventive agent) at high concentrations has an anti-oestrogenic effect. E2 and resveratrol are conjugated via common uridine diphosphoglucuronosyltransferase (UGT) and sulfotransferases (SULT) enzymes. Experiments were conducted in MCF-7 mammalian cells stably expressing human SULT1A1 or SULT1E1 to observe the effect of resveratrol on E2-mediated cell proliferation. The combination of E2 and resveratrol did have a proliferative effect in cells expressing SULT1E1, but not in those expressing SULT1A1. The effect of resveratrol (1-500 microM) on the glucuronidation of E2 (0.25-2.25 microM) was characterized in human liver microsomes. The highest resveratrol concentration significantly decreased the intrinsic clearance of E2 glucuronidation. The results corroborate the reported significant inhibition of SULT1E1-mediated E2 sulfation in vitro by resveratrol. Thus, resveratrol may interact with E2 in vivo by inhibiting its conjugation.

Genistein induction of human sulfotransferases in HepG2 and Caco-2 cells.

Sulfotransferases are phase II drug-metabolizing enzymes. While the induction of sulfotransferases by hormones and endogenous molecules is relatively well known, induction by xenobiotics is not well studied. Isoflavones are naturally occurring phyto-oestrogens, mainly existing in soy food products. They have been described as health-promoting, disease-preventing dietary supplements and as agents with cancer-preventive activities. Recently, isoflavones have been reported to interact with nuclear receptors, including those that are known to mediate the induction of drug-metabolizing enzymes. In the present investigation, the isoflavone genistein was shown to be a xenobiotic inducer of human sulfotransferases in transformed human liver cells (HepG2) and colon carcinoma cells (Caco-2). Enzymatic activity assay, Western blot, and real-time reverse transcription-polymerase chain reaction (RT-PCR) results demonstrated that genistein significantly induced protein and mRNA expression of human simple phenol sulfotransferase (hSULT1A1) and human dehydroepiandrosterone sulfotransferase (hSULT2A1) in HepG2 and Caco-2 cells. The induction was time-dependent and dose-dependent. Western blot results agreed well with real-time RT-PCR results, suggesting that induction occurred at the gene transcription level. This isoflavone is the first nutritionally related phyto-oestrogen shown to induce human sulfotransferases in HepG2 and Caco-2 cells.

Cloning, expression and purification of arylsulfate sulfotransferase from Eubacterium A-44.

A gene (astA) encoding arylsulfate sulfotransferase (ASST), which transfers a sulfate group from phenolic sulfate esters to phenolic acceptors, was cloned from a Eubacterium A-44 genomic library. The probe (1.5 kb fragment) for the astA gene was prepared from the PCR product of the primers produced using two internal amino acid sequences of ASST, which had been purified from Eubacterium A-44. The astA gene was cloned into the pKF3 vector. Its sequence revealed a 1863 bp open reading frame (ORF) encoding a protein containing 620 amino acids with a secretary signal peptide, and showed 91% homology (identity) to Eubacterium rectale IIIH previously reported. The cloned astA gene was expressed under the T7 promoter of the expression vectors, pET-39b(+) and pET-26b(+), in Escherichia coli BL21 (DE3), and the expressed ASSTs were purified using His Bind column chromatography. The specific activities of the purified ASSTs were 25.6 micromol/min/mg and 37.1 micromol/min/mg, respectively.

Involvement of p38 MAPK and Nrf2 in phenolic acid-induced P-form phenol sulfotransferase expression in human hepatoma HepG2 cells.

Phenolic acids have significant biological and pharmacological properties and some have demonstrated remarkable ability to alter sulfate conjugation. However, the modulation mechanisms of phenolic acids on phenol sulfotransferase expression have not been described. In the present study, we investigated the effects of phenolic acids on the expression of the Phase II P-form of phenol sulfotransferase (PST-P) in human hepatoma HepG2 cells. RT-PCR and western blot data revealed that gallic acid induced increase in PST-P expression at the mRNA and protein levels, respectively. This induction was also marked by an increase in PST-P activity. Actinomycin D and cycloheximide inhibited gallic acid-responsive PST-P mRNA expression, indicating that gallic acid is a requirement for transcription and de novo protein synthesis. Transient transfection of HepG2 cells with a reporter plasmid of the upstream region of the human PST gene caused a significant increase in reporter gene activity after gallic acid exposure. Moreover, gallic acid increased the nuclear levels of Nrf2, a transcription factor governing antioxidant response element (ARE). Electrophoretic mobility shift assay showed increased binding of nuclear proteins to ARE consensus sequence after treatment with gallic acid. While investigating the signaling pathways responsible for PST-P induction, we observed that gallic acid activated the p38 mitogen-activated protein kinase (MAPK) pathway. SB203580, a specific inhibitor of p38 MAPK, abolished gallic acid-induced PST-P protein expression. Similarly, gallic acid also caused an accumulation of Nrf2. Moreover, the protective effects of gallic acid on tert-butyl hydroperoxide-induced toxicity was partially blocked by p38 MAPK and PST-P inhibitors, further demonstrating that gallic acid attenuates oxidative stress through a pathway that involves p38 MAPK and PST-P. These results indicate that gallic acid is a potent inducer of PST-P and that PST-P induction is responsible for the gallic acid-mediated cytoprotection against oxidative damage.

Identification of a novel zebrafish SULT1 cytosolic sulfotransferase: cloning, expression, characterization, and developmental expression study.

By searching the zebrafish expressed sequence tag database, we had identified two partial cDNA clones encoding the 5'- and 3'-regions of a putative cytosolic sulfotransferase (SULT). Using the reverse transcription-polymerase chain reaction (RT-PCR) technique, a full-length cDNA encoding this zebrafish SULT was amplified, cloned, and sequenced. Analysis of the sequence data revealed that this novel zebrafish SULT displays 49, 46, and 45% amino acid sequence identity to human SULT1A1, mouse SULT1D1, and rat SULT1C1. This zebrafish SULT therefore appears to belong to the SULT1 cytosolic SULT gene family. Recombinant zebrafish SULT (designated SULT1 isoform 4), expressed using the pGEX-2TK prokaryotic expression vector and purified from transformed Escherichia coli cells, migrated as a 35kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Among the endogenous compounds tested as substrates, the purified SULT1 isoform 4 displayed significant sulfating activities toward thyroid hormones, estrone, and dehydroepiandrosterone. The enzyme also showed activities toward a number of xenobiotics including some flavonoids, isoflavonoids, and other phenolic compounds, with a pH optimum at 7.0. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 28 and 37 degrees C. Among 10 divalent metal cations tested, Fe2+, Hg2+, Co2+, Zn2+, Cu2+, and Cd2+ exhibited dramatic inhibitory effects on the activity of the enzyme. Developmental expression study using RT-PCR revealed that the zebrafish SULT1 isoform 4 showed a low level of expression in the segmentation period during the embryonic development, which gradually increased to a high level of expression throughout the larval stage onto maturity.

Altered expression of the hormone- and xenobiotic-metabolizing sulfotransferase enzymes 1A2 and 1C1 in malignant breast tissue.

Cytosolic sulfotransferases (SULTs) catalyze the biotransformation of steroid hormones as well as drugs and environmental toxins. Mostly, sulfonation leads to an inactivation of parent compounds, although formation of more toxic and cancerogenic metabolites also occurs. To assess possible alterations in the SULT enzyme expression pattern between malignant and non-malignant tissue, we studied the presence of 9 SULT enzymes of family 1 and 2 by semi-quantitative RT-PCR. Forty-two specimens from ductal and lobular breast carcinomas, lymph node metastasis, mastopathy and normal breast tissue were derived from 29 patients. Substantial expression of SULT 1A1, 1A2, 1A3, 1B1, 1C1, 1E1, 2A1, 2B1a and 2B1b mRNAs was observed in malignant and non-malignant tissue, although the pattern of the individual SULTs varied between the patients, and SULT1C1 mRNA was present in a greater number of malignant than non-malignant tissues (p<0.05). A major finding was that unspliced SULT1A2 mRNA, containing the complete intron between exons 7 and 8, was found in 4 of 16 non-malignant specimens, but was undetectable in the 26 malignant samples investigated. Taken together, the presence of various SULT enzymes in normal, premalignant and malignant breast tissue suggests an important role of SULT-mediated biotransformation in the breast. While the increased expression of SULT1C1 in malignant tissue seems to reflect tumor dedifferentiation, our finding of unspliced SULT1A2 mRNA in non-malignant tissue offers additional aspects regarding the search for breast cancer risk factors.

Examination of human tissue cytosols for expression of sulfotransferase isoform 1A2 (SULT1A2) using a SULT1A2-specific antibody.

Sulfotransferase isoform 1A2 (SULT1A2) is a member of the cytosolic sulfotransferase family of phase II detoxification enzymes. Studies with recombinant enzymes have shown that SULT1A2 can catalyze the bioactivation of several procarcinogens, indicating a potential role in chemical carcinogenesis. However, previous studies have suggested that the SULT1A2 transcript has a splicing defect that might prevent it from becoming translated into protein; therefore, we sought to determine the expression of SULT1A2 in tissues. An antibody directed against a region of human SULT1A2 that differs from other known sulfotransferase isoforms was developed and used to screen a large number of cytosolic fractions from various tissues. Although the SULT1A2 antibody recognized recombinant SULT1A2 and did not cross-react with other SULT isoforms, the expression of SULT1A2 was not detected in any tissue examined. These studies suggest that if SULT1A2 is expressed as protein, the levels are very low and that SULT1A2 probably does not play a physiological role in chemical carcinogenesis.

Effects of culture with TNF-alpha, TGF-beta and insulin on sulphotransferase (SULT 1A1 and 1A3) activity in human colon and neuronal cell lines.

The aim of the study was to determine whether the expression of sulphotransferase enzymes could be affected by the presence of cytokines or peptide hormones. The effects of cytokines (TNF-alpha and TGF-beta) and insulin on sulphotransferase (SULT 1A1 and 1A3) activity were studied in a human neuronal cell line (SK-N-SH) and a human gastrointestinal tract cell line (HT-29). Cells were cultured with varying concentrations of TNF-alpha, TGF-beta or insulin for 24 h; the SULT 1A1 isoform in the 2 cell lines showed different optimal substrate concentrations. There were no direct effects of cytokines on enzyme activity. Culture with TNF-alpha increased activity of both SULT 1A1 and 1A3 in the HT-29 cells; TGF-beta also increased activities of both isoforms but to a lesser extent; insulin increased activity of SULT 1A1 only. The cytokines and insulin had relatively little effect on sulphotransferase activity in the neuronal cell line. These results suggest that, unlike neuronal cells, gastrointestinal cells may respond to physiological states by altering sulphotransferase activity. As certain substrates such as diet-derived heterocyclic amines are bioactivated by sulphation to produce carcinogenic metabolites this may be a factor in the increased incidence of colorectal cancer in patients with inflammatory bowel disease or diabetes.

Stress regulation of sulfotransferases in male rat liver.

Sulfotransferase (SULT) catalyzed sulfation is responsible for hormone regulation and xenobiotic detoxification. Induction of SULTs by various hormones has been reported. Stress regulation of SULTs has not been reported, however. Here we report that rat liver SULTs can be regulated by physical stress (forced running, EX) and chemical stress (the organophosphorus pesticide parathion, PS). Both EX and PS increased rat liver phenol-sulfating SULT1A1 and hydroxysteroid-sulfating SULT2A1 activities. The increase in SULT1A1 activity did not correlate with protein (Western blot) or mRNA (RT-PCR) results but correlated well with increased non-protein soluble thiols. This suggests a possible Cys modification mechanism for stress regulation of SULT1A1. In vitro studies on GSH/GSSG effects on SULT1A1 activity support this conclusion. In contrast, SULT2A1 activity following physical or chemical stress treatments correlated well with protein and mRNA levels. This suggests a stress regulation mechanism of SULT2A1 at the gene transcription level, possibly occurring via hormones.