Localisation of aryl sulfotransferase expression in human tissues using hybridisation histochemistry and immunohistochemistry

To date, the laboratory has cloned seven unique human sulfotransferases; five aryl sulfotransferases (HAST1, HAST2, HAST3, HAST4 and HAST4v), an estrogen sulfotransferase and a dehydroepiandrosterone sulfotransferase. The cellular distribution of human aryl sulfotransferases in human hepatic and extrahepatic tissues has been determined using the techniques of hybridization histochemistry and immunohistochemistry. Human aryl sulfotransferase expression was detected in liver, epithelial cells of the gastrointestinal mucosal layer, epithelial cells lining bronchioles and in mammary duct epithelial cells. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

Isolation and characterisation of a novel rabbit sulfotransferase isoform belonging to the SULT1A subfamily

Sulfotransferases (SULTs) catalyse the sulfonation of both endogenous and exogenous compounds including hormones, catecholamines. drugs and xenobiotics. While in most occasions, sulfonation is a detoxication pathway. in the case of certain drugs and carcinogens. it leads to metabolic activation. Since, the rabbit has been extensively used for both pharmacological and toxicological studies, the purpose of this study was to further characterise the sulfotransferase system of this animal. In the present study, a novel sulfotransferase isoform (GenBank Accession no. AF360872) was isolated from a rabbit liver cDNA lambdaZAP 11 library. The full-length sequence of the clone was 1138 bp long and contained a coding region of 888 bp encoding a cytosolic protein of 295 amino acids (deduced molecular weight 34,193 Da). The amino acid sequence of this novel SULT isoform showed >70% identity with members of the SULT1A subfamily of sulfotransferases from other species. Upon expression of the encoded rabbit sulfotransferase in Escherchia coli (E. coli), it was shown that the enzyme was capable of sulfonating both p-nitrophenot (K-m and V-max values of 0.15 muM and 897.5 nmol/min/mg protein. respectively) and dopamine (K-m and V-max values of 175.3 muM and 151.1 nmol/min/mg protein, respectively). Based on the sequence data obtained and substrate specificity, this new rabbit sulfotransferase was named rabSULT1A1. Immunoblotting was used to demonstrate that rabSULT1A1 protein is expressed in liver, duodenum, jejunum, ileum, colon and recturm. (C) 2002 Elsevier Science Ltd. All rights reserved.

Active site mutations and substrate inhibition in human sulfotransferase 1A1 and 1A3

Human SULT1A1 is primarily responsible for sulfonation of xenobiotics, including the activation of promutagens, and it has been implicated in several forms of cancer. Human SULT1A3 has been shown to be the major sulfotransferase that sulfonates dopamine. These two enzymes shares 93% amino acid sequence identity and have distinct but overlapping substrate preferences. The resolution of the crystal structures of these two enzymes has enabled us to elucidate the mechanisms controlling their substrate preferences and inhibition. The presence of two p-nitrophenol (pNP) molecules in the crystal structure of SULT1A1 was postulated to explain cooperativity at low and inhibition at high substrate concentrations, respectively. In SULT1A1, substrate inhibition occurs with pNP as the substrate but not with dopamine. For SULT1A3, substrate inhibition is found for dopamine but not with pNP. We investigated how substrate inhibition occurs in these two enzymes using molecular modeling, site-directed mutagenesis, and kinetic analysis. The results show that residue Phe-247 of SULT1A1, which interacts with both p-nitrophenol molecules in the active site, is important for substrate inhibition. Mutation of phenylalanine to leucine at this position in SULT1A1 results in substrate inhibition by dopamine. We also propose, based on modeling and kinetic studies, that substrate inhibition by dopamine in SULT1A3 is caused by binding of two dopamine molecules in the active site. © 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Human sulfotransferases and their role in chemical metabolism

Sulfonation is an important reaction in the metabolism of numerous xenobiotics, drugs, and endogenous compounds. A supergene family of enzymes called sulfotransferases (SULTs) catalyze this reaction. In most cases, the addition of a sulfonate moiety to a compound increases its water solubility and decreases its biological activity. However, many of these enzymes are also capable of bioactivating procarcinogens to reactive electrophiles. In humans three SULT families, SULT1, SULT2, and SULT4, have been identified that contain at least thirteen distinct members. SULTs have a wide tissue distribution and act as a major detoxification enzyme system in adult and the developing human fetus. Nine crystal structures of human cytosolic SULTs have now been determined, and together with site-directed mutagenesis experiments and molecular modeling, we are now beginning to understand the factors that govern distinct but overlapping substrate specificities. These studies have also provided insight into the enzyme kinetics and inhibition characteristics of these enzymes. The regulation of human SULTs remains as one of the least explored areas of research in the field, though there have been some recent advances on the molecular transcription mechanism controlling the individual SULT promoters. Interindividual variation in sulfonation capacity may be important in determining an individual's response to xenobiotics, and recent studies have begun to suggest roles for SULT polymorphism in disease susceptibility. This review aims to provide a summary of our present understanding of the function of human cytosolic sulfotransferases.

Allelic Variation Investigation of the Estrogen Receptor Within an Australian Multiple Sclerosis Population

Multiple Sclerosis (MS) is a central nervous system (CNS) chronic inflammatory demyelinating disease leading to various neurological disabilities. The disorder is more prevalent for women with a ratio of 3:2 female to male. Objectives: To investigate variation within the estrogen receptor 1 (ESR1) polymorphism gene in an Australian MS case-control population using two intragenic restriction fragment length polymorphisms; the G594A located in exon 8 detected with the BtgI restriction enzyme and T938C located in intron 1, detected with PvuII. One hundred and ten Australian MS patients were studied, with patients classified clinically as Relapsing Remitting MS (RR-MS), Secondary Progressive MS (SP-MS) or Primary Progressive MS (PP-MS). Also, 110 age, sex and ethnicity matched controls were investigated as a comparative group. No significant difference in the allelic distribution frequency was found between the case and control groups for the ESR1 PvuII (P = 0.50) and Btg1 (P = 0.45) marker. Our results do not support a role for these two ESR1 markers in multiple sclerosis susceptibility, however other markers within ESR1 should not be excluded for potential involvement in the disorder.

Effect of estrogen on vascular smooth muscle cells is dependent upon cellular phenotype

To investigate the growth-regulating action of estrogen on vascular smooth muscle cells (SMC), effects of beta-17-estradiol (beta-E-2) on phenotypic modulation and proliferation of rabbit aortic SMC were observed in vitro. At 10(-8) M, beta-E-2 significantly slowed the decrease in volume fraction of myofilaments (V(v)myo) of freshly dispersed SMCs in primary culture, indicating an inhibitory effect of beta-E-2 On spontaneous phenotypic modulation of SMC from a contractile to a synthetic phenotype. Freshly dispersed SMCs treated with beta-E-2 also had a relatively longer quiescent phase than control cells before intense proliferation occurred. This was in contrast to SMCs in passage 2-3 (synthetic state), where beta-E-2-treated cells replicated significantly faster than untreated cells. beta-E-2 also markedly enhanced the serum-induced DNA synthesis of synthetic SMCs in a concentration-dependent manner within physiological range (10(-10) to 10-8 M). These findings indicate that the growth-regulating effect of estrogen on vascular SMC is dependent on the cell's phenotypic stare. It delays the cell cycle re-entry of the contractile SMCs by retarding their phenotypic modulation however, once cells have modulated to the synthetic phenotype, it promotes their replication. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

Expression of the estrogen receptor-associated immunophilins, cyclophilin 40 and FKBP52, in breast cancer

The estrogen receptor alpha (ER alpha) is implicated in the development of breast cancer. The immunophilins, cyclophilin 40 (CyP40) and FKBP52, are associated with ER alpha and other steroid receptors in mutually exclusive heterocomplexes and may differentially modulate receptor activity. Since previous studies have not assessed the levels of these immunophilins in breast cancer, we examined 10 breast cancer cell lines for mRNA and protein expression of CyP40 and FKBP52 and for amplification of the CyP40 gene. In addition, 26 breast carcinomas, including seven with matched normal breast tissue, were examined for mRNA expression of both immunophilins. CyP40 and FKBP52 were ubiquitously expressed in breast cancer cell lines, but there were significant differences in their pattern of expression. FKBP52 protein levels were generally an order of magnitude greater than those for CyP40. FKBP52 mRNA expression correlated strongly with protein expression and was significantly higher in ER alpha-positive compared with ER alpha-negative cell lines. However, CyP40 mRNA expression did not correlate with protein expression, nor did expression of this immunophilin correlate with ER alpha status. Relatively high expression of CyP40 in one cell line (BT-20) could be attributed to amplification of the CyP40 gene. Both immunophilins were also ubiquitously expressed in breast carcinomas, and we demonstrate for the first time that both CyP40 and FKBP52 mRNA are overexpressed in breast tumors compared to matched normal breast controls. The overexpression of CyP40 and FKBP52, coupled with relative differences in their expression in tumors, may have important functional implications for ER alpha and other steroid receptors in breast cancer.

Allelic loss of cyclophilin 40, an estrogen receptor-associated immunophilin, in breast carcinomas

Purpose: Cyclophilin 40 (CyP40) is an estrogen receptor-associated protein which appears to modify receptor function. The aim of this study was to determine the extent of allelic loss at the CyP40 locus in a panel of breast carcinomas using a newly characterized microsatellite marker located upstream of the CyP40 gene and then to correlate this with losses at chromosomal sites for cancer-associated genes. Methods: Allelic loss at CyP40 was determined from patients' matched tumor and normal breast tissue using Genescan 672 software analysis of fluorescently labeled, PAGE-separated PCR products incorporating the marker. For each patient, allelic loss at CyP40 was then assessed and compared with losses at markers for various cancer-associated genes. Results: Allelic loss was detected in 30% of breast carcinomas from patients heterozygous for the CyP40 marker. All carcinomas demonstrating allelic loss were grade II or III invasive ductal carcinomas and generally showed multiple losses at other sites near known cancer-associated genes. Conclusions: The polymorphic marker which we characterized was useful in determining allelic loss at the CyP40 locus in breast cancer patients and when applied in these studies in conjunction with various cancer-associated gene markers, suggests that deletions in the region of the CyP40 gene might be a late event in breast tumor progression.

A Randomized Trial of Sodium Fluoride (60 mg) ± Estrogen in Postmenopausal Osteoporotic Vertebral Fractures

Postmenopausal Caucasian women aged less than 80 years (n = 99) with one or more atraumatic vertebral fracture and no hip fractures, were treated by cyclical administration of enteric coated sodium fluoride (NaF) or no NaF for 27 months, with precautions to prevent excessive stimulation of bone turnover. In the first study 65 women, unexposed to estrogen (-E study), age 70.8 +/- 0.8 years (mean SEM) were all treated with calcium (Ca) 1.0-1.2 g daily and ergocalciferol (D) 0.25 mg per 25 kg once weekly and were randomly assigned to cyclical NaF (6 months on. 3 months off, initial dose 60 mg/day; group F CaD, n = 34) or no NaF (group CaD, n = 3 1). In the second study 34 patients. age 65.5 +/- 1.2 years, on hormone replacement therapy (E) at baseline, had this standardized, and were all treated with Ca and D and similarly randomized (FE CaD, n = 17, E CaD, n = 17) (+E study). The patients were stratified according to E status and subsequently assigned randomly to NaF. Seventy-five patients completed the trial. Both groups treated with NaF showed an increase in lumbar spinal density (by DXA) above baseline by 27 months: FE CaD + 16.2% and F CaD +9.3% (both p = 0.0001). In neither group CaD nor E CaD did lumbar spinal density increase. Peripheral bone loss occurred at most sites in the F CaD group at 27 months: tibia/fibula shaft -7.3% (p = 0.005); femoral shaft -7.1% (p = 0.004); distal forearm -4.0% (p = 0.004); total hip -4.1% (p = 0. 003); and femoral neck -3.5% (p = 0.006). No significant loss occurred in group FE CaD. Differences between the two NaF groups were greatest at the total hip at 27 months but were not significant [p < 0.05; in view of the multiple bone mineral density (BMD) sites, an alpha of 0.01 was employed to denote significance in BMD changes throughout this paper]. Using Cox's proportional hazards model, in the -E study there were significantly more patients with first fresh vertebral fractures in those treated with NaF than in those not so treated (RR = 24.2, p = 0.008, 95% CI 2.3-255). Patients developing first fresh fractures in the first 9 months were markedly different between groups: -23% of F CaD, 0 of CaD, 29% of FE CaD and 0 of E CaD. The incidence of incomplete (stress) fractures was similar in the two NaF-treated groups. Complete nonvertebral fractures did not occur in the two +E groups, there were no differences between groups F CaD and CaD. Baseline BMD (spine and femoral neck) was related to incident vertebral fractures in the control groups (no NaF), but not in the two NaF groups. Our results and a literature review indicate that fluoride salts. if used, should be at low dosage, with pretreatment and co-treatment with a bone resorption inhibitor.

Localization of a brain sulfotransferase, SULT4A1, in the human and rat brain: An immunohistochemical study

Cytosolic sulfotransferases are believed to play a role in the neuromodulation of certain neurotransmitters and drugs. To date, four cytosolic sulfotransferases have been shown to be expressed in human brain. Recently, a novel human brain sulfotransferase has been identified and characterized, although its role and localization in the brain are unknown. Here we present the first immunohistochemical (IHC) localization of SULT4A1 in human brain using an affinity-purified polyclonal antibody raised against recombinant human SULT4A1. These results are supported and supplemented by the IHC localization of SULT4A1 in rat brain. In both human and rat brains, strong reactivity was found in several brain regions, including cerebral cortex, cerebellum, pituitary, and brainstem. Specific signal was entirely absent on sections for which preimmune serum from the corresponding animal, processed in the same way as the postimmune serum, was used in the primary screen. The findings from this study may assist in determining the physiological role of this SULT isoform.

The human sulfotransferase SULT1A1 gene is regulated in a synergistic manner by Sp1 and GA binding protein

Human sulfotransferase SULT1A1 is an important phase II xenobiotic metabolizing enzyme that is highly expressed in the liver and mediates the sulfonation of drugs, carcinogens, and steroids. Until this study, the transcriptional regulation of the SULT1A subfamily had been largely unexplored. Preliminary experiments in primary human hepatocytes showed that SULT1A mRNA levels were not changed in response to nuclear receptor activators, such as dexamethasone and 3-methylcolanthrene, unlike other metabolizing enzymes. Using HepG2 cells, the high activity of the TATA-less SULT1A1 promoter was shown to be dependent on the presence of Sp1 and Ets transcription factor binding sites (EBS), located within - 112 nucleotides from the transcriptional start site. The homologous promoter of the closely related SULT1A3 catecholamine sulfotransferase, which is expressed at negligible levels in the adult liver, displayed 70% less activity than SULT1A1. This was shown to be caused by a two-base pair difference in the EBS. The Ets transcription factor GA binding protein (GABP) was shown to bind the SULT1A1 EBS and could transactivate the SULT1A1 promoter in Drosophila melanogaster S2 cells. Cotransfection of Sp1 could synergistically enhance GABP-mediated activation by 10-fold. Although Sp1 and GABP alone could induce SULT1A3 promoter activity, the lack of the EBS on this promoter prevented a synergistic interaction between the two factors. This study reports the first insight into the transcriptional regulation of the SULT1A1 gene and identifies a crucial difference in regulation of the closely related SULT1A3 gene, which accounts for the two enzymes' differential expression patterns observed in the adult liver.