RESUMEN
Heparan sulfate (HS) proteoglycan chains are key components of the breast tumor microenvironment that critically influence the behavior of cancer cells. It is established that abnormal synthesis and processing of HS play a prominent role in tumorigenesis, albeit mechanisms remain mostly obscure. HS function is mainly controlled by sulfotransferases, and here we report a novel cellular and pathophysiological significance for the 3-O-sulfotransferase 3-OST3A (HS3ST3A), catalyzing the final maturation step of HS, in breast cancer. We show that 3-OST3A is epigenetically repressed in all breast cancer cell lines of a panel representative of distinct molecular subgroups, except in human epidermal growth factor receptor 2-positive (HER2+) sloan-kettering breast cancer (SKBR3) cells. Epigenetic mechanisms involved both DNA methylation and histone modifications, producing different repressive chromatin environments depending on the cell molecular signature. Gain and loss of function experiments by cDNA and siRNA transfection revealed profound effects of 3-OST3A expression on cell behavior including apoptosis, proliferation, response to trastuzumab in vitro and tumor growth in xenografted mice. 3-OST3A exerted dual activities acting as tumor-suppressor in lumA-michigan cancer foundation (MCF)-7 and triple negative-MD Anderson (MDA) metastatic breast (MB)-231 cells, or as an oncogenic factor in HER2+-SKBR3 cells. Mechanistically, fluorescence-resonance energy transfer-fluorescence-lifetime imaging microscopy experiments indicated that the effects of 3-OST3A in MCF-7 cells were mediated by altered interactions between HS and fibroblast growth factor-7 (FGF-7). Further, this interplay between HS and FGF-7 modulated downstream ERK, AKT and p38 cascades, suggesting that altering 3-O-sulfation affects FGFR2IIIb-mediated signaling. Corroborating our cellular data, a clinical study conducted in a cohort of breast cancer patients uncovered that, in HER2+ patients, high level expression of 3-OST3A in tumors was associated with reduced relapse-free survival. Our findings define 3-OST3A as a novel regulator of breast cancer pathogenicity, displaying tumor-suppressive or oncogenic activities in a cell- and tumor-dependent context, and demonstrate the clinical value of the HS-O-sulfotransferase 3-OST3A as a prognostic marker in HER2+ patients.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Receptor ErbB-2/genética , Sulfotransferasas/genética , Animales , Neoplasias de la Mama/patología , Metilación de ADN/genética , Femenino , Heparitina Sulfato/genética , Humanos , Células MCF-7 , Ratones , Pronóstico , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Both laboratory studies in healthy volunteers and clinical studies have suggested adverse interactions between antiplatelet drugs and other commonly used medications. Interactions described include those between aspirin and ibuprofen, aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs), and the thienopyridine, clopidogrel, and drugs inhibiting CYP2C19, notably the proton pump inhibitors (PPI) omeprazole and esomeprazole. Other interactions between thienopyridines and CYP3A4/5 have also been reported for statins and calcium channel blockers. The ibuprofen/aspirin interaction is thought to be caused by ibuprofen blocking the access of aspirin to platelet cyclo-oxygenase. The thienopyridine interactions are caused by inhibition of microsomal enzymes that metabolize these pro-drugs to their active metabolites. We review the evidence for these interactions, assess their clinical importance and suggest strategies of how to deal with them in clinical practice. We conclude that ibuprofen is likely to interact with aspirin and reduce its anti-platelet action particularly in those patients who take ibuprofen chronically. This interaction is of greater relevance to those patients at high cardiovascular risk. A sensible strategy is to advise users of aspirin to avoid chronic ibuprofen or to ingest aspirin at least 2 h prior to ibuprofen. Clearly the use of NSAIDs that do not interact in this way is preferred. For the clopidogrel CYP2C19 and CYP3A4/5 interactions, there is good evidence that these interactions occur. However, there is less good evidence to support the clinical importance of these interactions. Again, a reasonable strategy is to avoid the chronic use of drugs that inhibit CYP2C19, notably PPIs, in subjects taking clopidogrel and use high dose H2 antagonists instead. Finally, anti-platelet agents probably interact with other drugs that affect platelet function such as selective serotonin reuptake inhibitors, and clinicians should probably judge patients taking such combination therapies as at high risk for bleeding.
Asunto(s)
Inhibidores de Agregación Plaquetaria/efectos adversos , Antiinflamatorios no Esteroideos/efectos adversos , Aspirina/efectos adversos , Interacciones Farmacológicas , Medicina Basada en la Evidencia/métodos , Humanos , Farmacoepidemiología , Ensayos Clínicos Controlados Aleatorios como Asunto/métodosRESUMEN
Cultured cryopreserved human hepatocytes are extensively used as a model system for studying drug metabolism, although they remain poorly characterized in respect of the major conjugation reactions glucuronidation and sulfation. Using paracetamol (acetaminophen), we assessed eleven samples of cryopreserved human hepatocytes for their suitability to investigate the simultaneous glucuronidation and sulfation of xenobiotics and evaluated inhibitors of conjugation. Kinetic characterization showed broadly similar values for paracetamol conjugation by hepatocytes (as reported in the literature for in vitro systems), with Km values of approximately 6 mM and 0.3 mM for glucuronidation and sulfation, respectively. Substantial interindividual differences were observed. The hepatocytes demonstrated a strong dose-dependent switch from a preponderance of sulfation at low concentrations of paracetamol to glucuronidation at higher doses, consistent with routes of clearance in vivo. A number of drugs, some of which such as probenecid and sulfinpyrazone are known to interact with paracetamol in vivo, were demonstrated to inhibit the sulfation and/or glucuronidation of paracetamol in hepatocytes, demonstrating the potential application of this model system for studying drug-drug interactions involving conjugation.
Asunto(s)
Interacciones Farmacológicas , Glucurónidos/metabolismo , Hepatocitos/metabolismo , Preparaciones Farmacéuticas/metabolismo , Sulfatos/metabolismo , Acetaminofén/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Criopreservación , Glucurónidos/química , Hepatocitos/efectos de los fármacos , Humanos , Cinética , Modelos Biológicos , Sulfatos/químicaRESUMEN
1. The relative roles of various members of the human sulfotransferase (SULT) enzyme family in the metabolism of apomorphine, a dopamine receptor antagonist used in the treatment of Parkinson's disease and, more recently, erectile dysfunction, were examined. In humans, sulfation is the major route of metabolism of this drug. 2. Using recombinant SULTs expressed in Escherichia coli, R(--)-apomorphine sulfation was studied using the universal barium precipitation assay in the presence of [35S] 3'-phosphoadenosine 5'-phosphosulfate and SULTs 1A1, 1A2, 1A3, 1B1, 1C2, 1E1 and 2A1. It was shown that SULTs 1A1, 1A2, 1A3 and 1E1 all sulfated apomorphine to varying extents. Low activity with SULT1B1 was only seen at the highest concentration (100 microM) and no activity with SULT1C2 or SULT2A1 was observed. 3. Kinetic analysis using purified recombinant SULTs showed that 1A1, 1A3 and 1E1 all had similar Vmax/Km values, although SULT1E1 had a slightly lower Km at around 1 microM compared with approximately 4 microM for the other SULTs. 4. By correlating apomorphine sulfation (at 10 microM) in a bank of 28 liver cytosols with SULT activity towards 10 microM 4-nitrophenol (SULT1A1) and 0.2 microM 17beta-oestradiol (SULT1E1), a strong correlation with SULT1A1 activity was clearly demonstrated, suggesting this enzyme was primarily responsible for hepatic apomorphine sulfation. 5. These findings were confirmed using immuno-inhibition experiments with antibodies against SULT1A and SULT1E1, which showed preferential inhibition of apomorphine sulfation in human liver cytosol by anti-SULT1A. 6. The results strongly implicate SULT1A1 as the major enzyme responsible for hepatic apomorphine metabolism. As SULT1A1 is subject to a common functional polymorphism, sulfation phenotype may be an important determinant of susceptibility to side-effects of apomorphine and/or efficacy of treatment.
Asunto(s)
Apomorfina/metabolismo , Arilsulfotransferasa/metabolismo , Agonistas de Dopamina/metabolismo , Arilsulfotransferasa/antagonistas & inhibidores , Sistema Libre de Células , Citosol/enzimología , Citosol/metabolismo , Escherichia coli/metabolismo , Humanos , Cinética , Fenotipo , Sulfatos/metabolismoRESUMEN
Members of the cytosolic sulfotransferase (SULT) superfamily catalyse the sulfation of a multitude of xenobiotics, hormones and neurotransmitters. Humans have at least 10 functional SULT genes, and a number of recent advances reviewed here have furthered our understanding of SULT function. Analysis of expression patterns has shown that sulfotransferases are highly expressed in the fetus, and SULTs may in fact be a major detoxification enzyme system in the developing human. The X-ray crystal structures of three SULTs have been solved and combined with mutagenesis experiments and molecular modelling, they have provided the first clues as to the factors that govern the unique substrate specificities of some of these enzymes. In the future these and other studies will facilitate prediction of the fate of chemicals metabolised by sulfation. Variation in sulfation capacity may be important in determining an individual's response to xenobiotics, and there has been an explosion in information on sulfotransferase polymorphisms and their functional consequences, including the influence of SULT1A1 genotype on susceptibility to colorectal and breast cancer. Finally, the first gene knockout experiments with SULTs have recently been described, with the generation of estrogen sulfotransferase deficient mice in which reproductive capacity is compromised. Our improved understanding of these enzymes will have significant benefits in such diverse areas as drug design and development, cancer susceptibility, reproduction and development.
Asunto(s)
Farmacogenética/tendencias , Sulfotransferasas/genética , Sulfotransferasas/metabolismo , Tecnología Farmacéutica/tendencias , Animales , Humanos , Farmacogenética/métodos , Sulfotransferasas/química , Tecnología Farmacéutica/métodosRESUMEN
Thyroid hormone is essential for normal human development, and disruption of thyroid hormone homeostasis at critical developmental stages can result in severe and often long-term effects on crucial organs such as the brain and lungs. Numerous factors control the bioavailability of receptor active thyroid hormone T(3). Sulfation, catalyzed by sulfotransferase enzymes (SULTs), is an important pathway of thyroid hormone metabolism by which T(4) is irreversibly converted to inactive reverse T(3) rather than active T(3). The human fetus and neonate have high levels of circulating sulfated iodothyronines, although the source of these is not clear. The placenta forms the link between the fetus and its mother and is involved in transfer of thyroid hormone early in pregnancy, although its capacity for sulfation is unknown. We therefore examined expression of the SULTs involved in iodothyronine metabolism during human placental development. SULT activity was measured in human placental cotyledon and membranes (amnion, chorion, and decidua basalis) from 13-42 wk of gestation, and Western blot analysis was employed to verify enzyme activity data. Phenol and catecholamine sulfotransferases were expressed at the highest levels and were generally higher in the villous than membranous tissues. SULT1A1 activity showed significant correlation with sulfation of 3,3'-T(2), suggesting that this enzyme is primarily responsible for placental T(2) sulfation. Estrogen sulfotransferase was present at extremely low levels during early pregnancy, although in mid- and late gestation increased expression in the (predominantly maternal-derived) decidual component of the placenta was observed. Hydroxysteroid sulfotransferase, T(3), reverse T(3), and T(4) SULT activities were also low in all tissues examined, and expression of SULTs 1B1 and 1C2 were essentially undetectable by Western blot analysis. The results highlight a tissue-specific regulation of SULT expression during placental development, demonstrate very low sulfation of iodothyronines suggesting that the placenta is not a major source of circulating sulfated iodothyronines in the fetus.
Asunto(s)
Placenta/enzimología , Placentación , Sulfotransferasas/fisiología , Hormonas Tiroideas/metabolismo , Femenino , Humanos , Immunoblotting , Inmunohistoquímica , Isoenzimas/metabolismo , Embarazo , Sulfotransferasas/metabolismo , Distribución TisularRESUMEN
Excessive glucose production by the liver contributes significantly to diabetic hyperglycemia. The enzyme system glucose-6-phosphatase plays a key role in regulating hepatic glucose production and therefore its inhibition is a potential therapeutic target for the correction of hyperglycemia. It has previously been shown that sulfated steroids, such as estrone sulfate and dehydroepiandrosterone sulfate, inhibit the glucose-6-phosphatase system in vitro, principally through inhibition of endoplasmic reticulum glucose-6-phosphate transport. We report here that in the obese/diabetic ob/ob mouse model, orally administered estrone sulfate reduces the abnormally elevated hepatic glucose-6-phosphatase enzyme activity and enzyme protein levels that are characteristic in the ob/ob mouse, and that this reduction is associated with normalization of blood glucose levels. Other sulfated and non-sulfated steroids also reduced, to a lesser extent, glucose-6-phosphatase enzyme activity - with the exception of dehydroepiandrosterone sulfate, which had no apparent effect on this system in ob/ob mice. Estrone sulfate is therefore an effective antihyperglycemic agent in ob/ob mice, and the glucose-6-phosphatase system can be successfully targeted for the therapeutic management of hyperglycemia in this animal model of non-insulin-dependent diabetes mellitus.
Asunto(s)
Diabetes Mellitus/tratamiento farmacológico , Estradiol/análogos & derivados , Estrona/análogos & derivados , Estrona/uso terapéutico , Glucosa-6-Fosfatasa/metabolismo , Hipoglucemiantes/uso terapéutico , Hígado/enzimología , Obesidad , Envejecimiento , Animales , Glucemia/análisis , Sulfato de Deshidroepiandrosterona/farmacología , Diabetes Mellitus/enzimología , Diabetes Mellitus/genética , Estradiol/farmacología , Estrona/farmacología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Microsomas Hepáticos/enzimologíaRESUMEN
Sulphation is an important detoxification pathway for numerous xenobiotics; however, it also plays an important role in the metabolism and bioactivation of many dietary and environmental mutagens, including heterocyclic amines implicated in the pathogenesis of colorectal and other cancers. A major sulphotransferase (SULT) enzyme in humans, SULT1A1, is polymorphic with the most common variant allele, SULT1A1*2, occurring at a frequency of about 32% in the Caucasian population. This allele codes for an allozyme with low enzyme activity and stability compared to the wild-type (SULT1A1*1) enzyme, and therefore SULT1A1 genotype may influence susceptibility to mutagenicity following exposure to heterocyclic amines and other environmental toxins. Previously, a significant association of SULT1A1*1 genotype with old age has been observed, suggesting a 'chemoprotective' role for the high-activity phenotype. Here we have compared the frequencies of the most common SULT1A1 alleles in 226 colorectal cancer patients and 293 previously described control patients. We also assessed whether SULT1A1 genotype was related to various clinical parameters in the patient group, including Duke's classification, differentiation, site, nodal involvement and survival. There was no significant difference in allele frequency between the control and cancer patient populations, nor was there a significant association with any of the clinical parameters studied. However, when the age-related difference in allele frequency was considered, a significantly reduced risk of colorectal cancer (odds ratio = 0.47; 95% confidence interval = 0.27-0.83; P = 0.009), was associated with homozygosity for SULT1A1*1 in subjects under the age of 80 years. These results suggest that the high activity SULT1A1*1 allozyme protects against dietary and/or environmental chemicals involved in the pathogenesis of colorectal cancer.
Asunto(s)
Alelos , Arilsulfotransferasa , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/genética , Sulfotransferasas/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Colorrectales/epidemiología , Femenino , Pruebas Genéticas , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo Genético/genética , Factores de Riesgo , Factores SexualesRESUMEN
Sulfation is an important mechanism for regulating the biological activity of numerous hormones and neurotransmitters in man. Here we have investigated the ontogeny of sulfotransferases (SULT) and sulfatase (ARS) involved in the metabolism of thyroid hormone and dopamine. SULT1A1 enzyme activity was lower in postnatal liver and lung than in fetal tissues. Hepatic SULT1A3 (dopamine) was expressed at high levels early in development, but decreased substantially in late fetal/early neonatal liver and was essentially absent from the adult liver. In lung, significant SULT1A3 activity was observed in the fetus, but neonatal levels were considerably lower. In brain, the highest activity was observed in the choroid plexus for SULT1A1, with low and widespread activity for both SULT1A1 and SULT1A3 in other brain regions. SULT activity with 3,3'-diiodothyronine (3,3'-T(2)) as substrate was measured in all tissues and correlated significantly with SULT1A1 activity (4-nitrophenol), suggesting that SULT1A1 is primarily responsible for the sulfation of this iodothyronine. The developmental expression of SULT1A3 and SULT1A1 in liver and brain was confirmed by immunoblot, and immunohistochemistry of developing liver showed substantial expression of these proteins in hemopoietic cells in fetal liver. We also detected low activity for the hydrolysis of 3,3'-T(2) sulfate by ARS, although there was less distinction between fetal and neonatal samples than with SULT activities. We have therefore shown that the developing fetus has substantial sulfation capacity. Sulfation may therefore play a major role in the homeostasis of hormones and other endogenous compounds as well as in detoxification in the fetus, particularly as other conjugating enzyme systems, such as the UDP-glucuronosyltransferases, are not expressed at significant levels until the neonatal period.
Asunto(s)
Envejecimiento/metabolismo , Dopamina/metabolismo , Feto/metabolismo , Sulfatos/metabolismo , Hormonas Tiroideas/metabolismo , Arilsulfatasas/metabolismo , Arilsulfotransferasa/metabolismo , Encéfalo/enzimología , Cadáver , Preescolar , Diyodotironinas/metabolismo , Humanos , Immunoblotting , Inmunohistoquímica , Lactante , Recién Nacido , Hígado/enzimología , Pulmón/enzimología , Sulfotransferasas/metabolismoRESUMEN
Thyroid hormones are involved in the regulation of fetal lung development, and maturation is accelerated in animal models by antepartum exposure to raised concentrations of the receptor-active thyroid hormone triiodothyronine and glucocorticoids. It is essential that the nature of the regulation of the spatial and temporal metabolism of iodothyronines in the human fetus and infant is known before effective therapies can be developed to modify human lung maturation. Thyroid hormone bioavailability to the human fetus is regulated in part by enzymatic deiodination and reversible sulfation of iodothyronines, with contributions from other factors such as fetomaternal and fetoamniotic hormone transfers, fetal thyroid gland production, and the activities of plasma membrane transporters mediating uptake of iodothyronines from plasma into tissues.
Asunto(s)
Pulmón/embriología , Pulmón/metabolismo , Hormonas Tiroideas/metabolismo , Arilsulfatasas/metabolismo , Femenino , Madurez de los Órganos Fetales , Humanos , Yoduro Peroxidasa/metabolismo , Embarazo , Sulfatos/metabolismo , Sulfotransferasas/metabolismo , Hormonas Tiroideas/farmacologíaRESUMEN
The expression of sulfotransferase and steroid sulfatase was studied in rat liver using the most promising culture models of hepatocytes, including monolayer culture with a pyruvate (30 mM) enriched medium, co-culture with rat epithelial cells from primitive biliary origin and collagengel sandwich culture. In the latter, addition of dexamethasone (1 microM) to the medium was examined. Phenol sulfotransferase enzymes (SULT1) were studied by measuring activities towards 4-methylphenol and estradiol, hydroxysteroid sulfotransferase (SULT2A) activity was determined towards dehydroepiandrosterone (DHEA). Microsomal steroid sulfatase activity was measured towards estrone sulfate. Western blot analysis was carried out using polyclonal antibodies raised against rat phenol sulfotransferase SULT1A1 (ASTIV), estrogen sulfotransferase SULT1E1 (EST) and hydroxysteroid sulfotransferase (HST). SULT2A activity towards DHEA was maintained at a high level during the whole culture time. In the co-culture it even reached the level of freshly isolated cells. Addition of pyruvate had no positive effect on the activity measured in monolayer cultures. High SULT1A1 activity towards 4-methylphenol was found in the co-culture system. In the monolayer culture, the activity initially decreased with 35% but was then kept at a constant level, while in the sandwich culture low activities were measured. For dexamethasone, an inducing effect on the various SULT activities could not be detected. Independently of the culture model used, the SULT1E1 activity towards estradiol decreased to 20% and 5% of the initial activity after four and seven days of culture, respectively. Microsomal steroid sulfatase activity was best maintained in collagengel sandwich cultures. During the first four days in culture it retained 73% of the initial activity, afterwards it decreased to 40% of the activity found in freshly isolated hepatocytes, irrespective of the culture conditions. High expectations exist for collagengel sandwich cultures, however, in our study the results were rather disappointing. Monolayer is a suitable culture model for short-term purposes. For long-term in vitro biotransformation studies, co-culture is preferred but is rather complex.
Asunto(s)
Arilsulfotransferasa , Hepatocitos/enzimología , Sulfatasas/metabolismo , Sulfotransferasas/metabolismo , Albúminas/metabolismo , Animales , Células Cultivadas , Cresoles/metabolismo , Medios de Cultivo/farmacología , Deshidroepiandrosterona/metabolismo , Estradiol/metabolismo , Hepatocitos/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Sulfation plays a major role in the detoxication of xenobiotics as well as in modulating the biological activity of numerous important endogenous chemicals. In contrast to this "chemical defense" function, sulfation is also a key step in the bioactivation of a host of pro-mutagens and pro-carcinogens. These reactions are catalyzed by an expanding family of sulfotransferase (SULT) enzymes, which transfer a sulfuryl moiety from the universal donor 3'-phosphoadenosine 5'-phosphosulfate. Here, we discuss current knowledge of the human sulfotransferase enzyme family, of which at least 11 members have been identified to date, including regulation of expression by endogenous compounds and xenobiotics as well as the molecular basis of polymorphisms in members of the SULT1A (phenol sulfotransferase) family. We also present new data on the inhibition of SULT1A enzymes by dietary chemicals, showing that compounds to which we are exposed regularly, such as epigallocatechin gallate and epicatechin gallate are extremely potent inhibitors of phenol sulfotransferases (K(i) in the nanomolar range for SULT1A1). We found that the mechanism of inhibition by these chemicals varied depending on the individual isoform involved, showing uncompetitive inhibition of SULT1A1 whereas with SULT1A2 and -1A3 they demonstrated mixed type inhibition. Thus, genetic-environmental interactions may play an important role in modulating sulfotransferase activity and in determining individual response to chemicals metabolized by these important enzymes.
Asunto(s)
Dieta , Sulfotransferasas/metabolismo , Biotransformación , Carcinógenos/farmacocinética , Carcinógenos/farmacología , Regulación Enzimológica de la Expresión Génica , Humanos , Mutágenos/farmacocinética , Mutágenos/farmacología , Polimorfismo Genético , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Sulfotransferasas/antagonistas & inhibidores , Sulfotransferasas/genéticaRESUMEN
Polychlorinated biphenyls (PCBs) are persistent environmental pollutants which exert a variety of toxic effects in animals, including disturbances of sexual development and reproductive function. The estrogenic effects of PCBs may be mediated in part by hydroxylated PCB metabolites (PCB-OHs), but the mechanisms by which they are brought about are not understood. PCBs as well as PCB-Hs show low affinities for both alpha and beta estrogen receptor isoforms. In the present study we demonstrate that various environmentally relevant PCB-OHs are extremely potent inhibitors of human estrogen sulfotransferase, strongly suggesting that they indirectly induce estrogenic activity by increasing estradiol bioavailability in target tissues.
Asunto(s)
Contaminantes Ambientales/farmacología , Bifenilos Policlorados/farmacología , Sulfotransferasas/antagonistas & inhibidores , Disponibilidad Biológica , Estradiol/farmacocinética , Humanos , Hidroxilación , Técnicas In Vitro , CinéticaRESUMEN
Dehydroepiandrosterone sulfotransferase (DHEA-ST) is a key enzyme in the formation of Dehydroepiandrosterone sulfate (DHEAS) and is thought to be involved in the conversion of various substances such as bile acids and cholesterol. The existence of DHEA-ST in the small intestine in addition to the adrenal gland and liver in adult humans was recently reported. As the sulfotransferases can act on toxic or potentially toxic substances to reduce their biological activity, we attempted to clarify the significance of DHEA-ST in gastrointestinal tract. We examined surgically resected human stomach for the presence of DHEA-ST and attempted to determine its possible biological significance. DHEA-ST activity ranged widely from 6 to 84 pmoles/mg protein/90 min in 7 cases. Immunoblotting revealed one single band of a 35-kDa protein corresponding to the moleculr weight of DHEA-ST. Both DHEA-ST immunoreactivity and mRNA hybridization signals were localized in parietal cells of the gastric glands. The results of our present study demonstrated that the sulfation of DHEA by DHEA-ST occurs in the gastric glands. The localization of DHEA-ST in parietal cells suggests that this enzyme is correlated to mucosal function in the human stomach in addition to detoxification of exogenous substances.
Asunto(s)
Mucosa Gástrica/enzimología , Sulfotransferasas/genética , Sulfotransferasas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Sondas de ADN/genética , Femenino , Expresión Génica , Humanos , Immunoblotting , Inmunohistoquímica , Hibridación in Situ , Inactivación Metabólica , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The mammalian xenobiotic-metabolizing sulfotransferases are cytosolic enzymes, which form a gene superfamily (SULT). Ten distinct human SULT forms are known. Two SULT forms represent splice variants, the other forms are encoded by separate genes. Common functional polymorphisms of the transcribed region are known for two of the forms. We have expressed 16 separate rat and human SULTs as well as some of their allelic variants, in Salmonella typhimurium TA1538 and/or V79 cells, which are target cells of commonly used mutagenicity assays. The expressed SULTs activated numerous compounds to mutagens in both assay systems. However, some promutagens were activated by only one or several of the human SULTs. Pronounced differences in promutagen activation were also detected between orthologous rat and human SULTs, and between allelic variants of human SULTs.
Asunto(s)
Mutágenos/toxicidad , Sulfotransferasas , Animales , Clonación Molecular , Variación Genética , Humanos , Pruebas de Mutagenicidad , Polimorfismo Genético , Ratas , Salmonella typhimurium , Sulfotransferasas/clasificación , Sulfotransferasas/efectos de los fármacos , Sulfotransferasas/genética , Sulfotransferasas/fisiología , ToxicologíaRESUMEN
The industrial solvent 2-nitropropane (2-NP) is a genotoxic hepatocarcinogen in rats. The genotoxicity of the compound in rats has been attributed to sulfotransferase-mediated formation of DNA-reactive nitrenium ions from the anionic form of 2-NP, propane 2-nitronate (P2N). Whether human sulfotransferases are capable of activating P2N is unknown. In the present study we have addressed this question by investigating the genotoxicity of P2N in various V79-derived cell lines engineered for expression of individual forms of human sulfotransferases, the phenol-sulfating and the monoamine-sulfating phenol sulfotransferases (hP-PST and hM-PST) and the human hydroxysteroid sulfotransferase (hHST). Genotoxicity was assessed by measuring the induction of DNA repair synthesis and by analyzing the formation of DNA modifications. P2N induced repair synthesis in V79-hP-PST and V79-hM-PST cells, whereas induction of repair synthesis in V79-hHST cells was negligible. P2N also resulted in the formation of 8-aminodeoxyguanosine and increased the level of 8-oxodeoxyguanosine in V79-hP-PST cells, but not in the parental V79-MZ cells, which do not show any sulfotransferase activity. Acetone oxime, the tautomeric form of the first reduction product of 2-NP, 2-nitrosopropane, was inactive in all cell lines. The results show that the human phenol sulfotransferases P-PST and M-PST are capable of metabolically activating P2N (P-PST >> M-PST) and that the underlying mechanism is apparently identical to that resulting in the activation of P2N in rat liver, where 2-NP causes carcinomas. These results support the notion that 2-NP should be regarded as a potential human carcinogen.
Asunto(s)
Arilsulfotransferasa , Carcinógenos/farmacocinética , Nitroparafinas/metabolismo , Nitroparafinas/farmacocinética , Profármacos/farmacocinética , Propano/análogos & derivados , Sulfotransferasas/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Biotransformación , Línea Celular , Cricetinae , Cricetulus , Aductos de ADN , Daño del ADN , Reparación del ADN/efectos de los fármacos , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análisis , Humanos , Pulmón/citología , Pruebas de Mutagenicidad , Oximas/metabolismo , Propano/metabolismo , Propano/farmacocinética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The endometrium plays a key role in reproduction, and this function is tightly regulated by endogenous and xenobiotic steroids. Sulphation, catalysed by members of the sulphotransferase (SULT) enzyme family, is a major deactivating mechanism for steroid hormones and we have investigated the expression and regulation in vivo of SULT in the human endometrium. In the normal cycling endometrium, expression of the phenol sulphotransferases SULT1A1 and SULT1A3 and the oestrogen sulphotransferase SULT1E1 were observed, with SULT1A1 and SULT1E1 expression being higher in the luteal phase than in the follicular phase. No expression of the hydroxysteroid sulphotransferase SULT2A1 was detected at any time in the endometrium. In endometrium from women taking the combined oral contraceptive pill (OCP), SULT1E1 expression was virtually absent, and SULT1A1 expression was substantially reduced. Similarly, in early pregnancy (i.e. first trimester) endometrium, SULT1E1 expression was absent, although SULT1A1 and SULT1A3 expression were unaffected. Our results with normal endometrium support in-vitro data showing that SULT1E1 expression is regulated by progesterone. However, the data obtained from OCP and early pregnancy endometrium suggest that factors other than the concentration of circulating progesterone are involved in the regulation of the expression of this important enzyme in the endometrium.
Asunto(s)
Anticonceptivos Orales/farmacología , Endometrio/enzimología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Ciclo Menstrual/genética , Sulfotransferasas/genética , Adulto , Interacciones Farmacológicas , Femenino , Humanos , Persona de Mediana Edad , Embarazo , Sulfotransferasas/biosíntesisRESUMEN
Humans are one of the few species that produce large amounts of catecholamine sulfates, and they have evolved a specific sulfotransferase, SULT1A3 (M-PST), to catalyze the formation of these conjugates. An orthologous protein has yet to be found in other species. To further our understanding of the molecular basis for the unique substrate selectivity of this enzyme, we have solved the crystal structure of human SULT1A3, complexed with 3'-phosphoadenosine 5'-phosphate (PAP), at 2.5 A resolution and carried out quantitative structure-activity relationship (QSAR) analysis with a series of phenols and catechols. SULT1A3 adopts a similar fold to mouse estrogen sulfotransferase, with a central five-stranded beta-sheet surrounded by alpha-helices. SULT1A3 is a dimer in solution but crystallized with a monomer in the asymmetric unit of the cell, although dimer interfaces were formed by interaction across crystallographic 2-fold axes. QSAR analysis revealed that the enzyme is highly selective for catechols, and catecholamines in particular, and that hydrogen bonding groups and lipophilicity (cLogD) strongly influenced K(m). We also investigated further the role of Glu(146) in SULT1A3 using site-directed mutagenesis and showed that it plays a key role not only in defining selectivity for dopamine but also in preventing many phenolic xenobiotics from binding to the enzyme.
Asunto(s)
Arilsulfotransferasa/química , Alanina/química , Sustitución de Aminoácidos , Arilsulfotransferasa/metabolismo , Cristalografía por Rayos X , Dimerización , Ácido Glutámico/química , Humanos , Cinética , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The importance of estrogens in bone metabolism is illustrated by the accelerated bone loss and increase in osteoporotic fractures associated with postmenopausal estrogen deficiency. In this study, the expression and activity of the enzymes involved in estrogen metabolism in human osteoblastic cells were investigated in relation to differentiation of these cells. PCR reactions using mRNA from an in vitro differentiating human cell line (SV-HFO) were performed to assess mRNA expression of the enzymes aromatase, different subtypes of 17beta-hydroxysteroid dehydrogenase (17beta-HSD), and steroid sulfatase. Aromatase, sulfatase, and 17beta-HSD type 2 and 4 were found to be expressed throughout differentiation. Expression of 17beta-HSD type 3, however, was relatively weak, except for early time points in differentiation. Type 1 17beta-HSD expression was not detected. Aromatase activity decreased during differentiation, as was demonstrated by the conversion of androstenedione (A) and testosterone (T) into estrone (E(1)) and estradiol (E(2)), respectively. The 17beta-HSD isozymes catalysing a reductive reaction convert androstenedione and estrone into testosterone and estradiol, respectively. Their activity declined with differentiation. Analysis of 17beta-HSD activity indicated both oxidative (E(2) to E(1); T to A) and reductive (E(1) to E(2); A to T) metabolism at all stages of osteoblast differentiation. Both activities declined as cells moved toward a differentiating mineralizing phenotype. However, the oxidative reaction was increasingly in favor of the reductive reaction at all times during differentiation. Sulfatase activity, as demonstrated by the conversion of estrone-sulfate into estrone, was constant during differentiation. In conclusion, we have demonstrated that all enzymes necessary for estrogen metabolism are expressed and biologically active in differentiating human osteoblasts. The activity of aromatase and 17beta-HSD was found to be dependent on the stage of cell differentiation. In addition, human osteoblasts effectively convert estradiol into estrone. The efficacy of osteoblasts to synthesize estradiol may determine the ultimate change in rate of bone turnover after menopause, as well as the development of osteoporosis. Moreover, the enzymes involved in the metabolism of estradiol may form a target for intervention.