Thermostable Phenol Sulfotransferase Research Articles

A Mixture‐of‐Genotypes Model for the Distribution of Thermostable Phenol Sulfotransferase Activity

AbstractA statistical method for parametric density estimation based upon a mixture‐of‐genotypes model is developed for the thermostable phenol sulfotransferase (SULT1A1) activity which has a putative role in modifying risk for colon and prostate cancer/polyps. The EM algorithm for the general mixture model is modified to accommodate the genetic constraints and is used to estimate genotype frequencies from the distribution of the SULT1A1 phenotype. A parametric bootstrap likelihood ratio test is considered as a testing method for the number of mixing components. The size and power of the test is then investigated and compared with the conventional chi‐squared test. The relative risk associated with genotypes defined by this model is also investigated through the generalized linear model. This analysis revealed that a genotype with the highest mean value of SULT1A1 activity has greater impact on cancer risk than others. This result suggests that the phenotype with a higher SULT1A1 activity might be important in studying the association between the cancer risk and SULT1A1 activity. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Phytoestrogens are potent inhibitors of estrogen sulfation: implications for breast cancer risk and treatment.

We investigated the ability of 37 flavonoids and flavonoid sulfoconjugates, including some abundant dietary constituents, to act as substrates and/or inhibitors of the sulfotransferase and sulfatase enzymes that interconvert active estrogens and inactive estrogen sulfates in human tissues. The enzymes studied include estrogen sulfotransferase, the thermostable phenolsulfotransferase that acts on a range of substrates including estrogens; steroid sulfatase; and two related enzymes, monoamine phenolsulfotransferase and arylsulfatase A. Several dietary flavonoids, including the soy isoflavones genistein and daidzein, were sulfated by these human sulfotransferases. Many flavonoids were potent inhibitors of thermostable phenolsulfotransferase. Genistein and equol were potent mixed inhibitors of hepatic estrogen sulfotransferase, with inhibitory constant values of 500 nM and 400 nM, respectively. Monoamine phenolsulfotransferase activity was relatively unaffected by flavonoids, but this enzyme was mainly responsible for the sulfation of flavonoids at concentrations greater than 1 micro M. Of the compounds tested, only daidzein 4,7-bisulfate, a trace metabolite in humans, significantly inhibited steroid sulfatase in the micromolar concentration range. Hence, dietary flavonoids may be able to influence the bioavailability of endogenous estrogens, and disrupt endocrine balance, by increasing the ratio of active estrogens to inactive estrogen sulfates in human tissues.

Sulfation of bisphenol A abolished its estrogenicity based on proliferation and gene expression in human breast cancer MCF-7 cells

Bisphenol A, an endocrine-disrupting chemical, is widely used in many consumer products. We previously showed the sulfoconjugation of bisphenol A catalyzed by a human thermostable phenol sulfotransferase, ST1A3. The estrogenic potency of bisphenol A sulfate was compared with that of bisphenol A by an E-screen assay using human breast cancer MCF-7 cells. An increase in the expression level of an estrogen-responsive pS2 gene was also examined using MCF-7 cells after exposure to bisphenol A and its sulfate for their estrogenicity. Bisphenol A sulfate did not exhibit estrogenic effects at 0.1 μ m (E-screen assay) and 1 m m (pS2 gene expression) compared with bisphenol A, which exhibited the effects at 3 n m (E-screen assay) and 1 μ m (pS2 gene expression), respectively. We have therefore evaluated major roles of cytosolic phenol sulfotransferase in the human liver. Bisphenol A sulfation in human liver cytosols was inhibited by more than 90% by p-nitrophenol and quercetin, a typical substrate and specific inhibitor of phenol sulfotransferase, respectively. These results indicated that the estrogenicity of bisphenol A was abolished through its sulfation catalyzed by a human hepatic thermostable phenol sulfotransferase.

Characterization of human liver thermostable phenol sulfotransferase (SULT1A1) allozymes with 3,3',5-triiodothyronine as the substrate.

Sulfotransferase 1A1 (SULT1A1) (thermostable phenol sulfotransferase, TS PST1, P-PST) is important in the metabolism of thyroid hormones. SULT1A1 isolated from human platelets displays wide individual variations not only in the levels of activity, but also in thermal stability. The activity of the allelic variant or allozyme SULT1A1*1, which possesses an arginine at amino acid position 213 (Arg213) has been shown to be more thermostable than the activity of the SULT1A1*2 allozyme which possesses a histidine at this position (His213) when using p-nitrophenol as the substrate. We isolated a SULT1A1*1 cDNA from a human liver cDNA library and expressed both SULT1A1*1 and SULT1A1*2 in eukaryotic cells. The allozymes were assayed using iodothyronines as the substrates and their biochemical properties were compared. SULT1A1*1 activity was more thermostable and more sensitive to NaCl than was SULT1A1*2 activity when assayed with 3,5,3'-triiodothyronine (T(3)). Sensitivities to 2,6-dichloro-4-nitrophenol (DCNP) and apparent K(m) values for SULT1A1*1 and for SULT1A1*2 with iodothyronines were similar. Based on K(m) values, the preferences of these SULT1A1 allozymes for iodothyronine substrates were the same (3,3'-diiodothyronine (3,3'-T(2))>3', 5',3-triiodothyronine (rT(3))>T(3)>thyroxine (T(4))>>3,5-diiodothyronine (3,5-T(2))). SULT1A1*1 activity was significantly higher than the SULT1A1*2 activity with T(3) as the substrate. Potential differences in thyroid hormone sulfation between individuals with predominant SULT1A1*1 versus SULT1A1*2 allozymes are most likely due to differences in catalytic activity rather than substrate specificity.

In vivo human metabolism of [2- 14C]2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP)

To better understand the interactions of the pathways of activation and detoxification on the metabolism of the putative carcinogen, PhIP, we administered a dose of 70–84 μg [2- 14C] PhIP (17.5 μCi 14C) 48–72 h before scheduled colon surgery. Blood and urine collected for the next 48–72 h was evaluated by linear accelerator mass spectroscopy (AMS) and scintillation counting LC-MS to identify specific PhIP metabolites. The thermostable phenol sulfotransferase (SULT1A1) phenotype was correlated with the 4′-PhIP-SO 4 levels in the urine at 0–4 h ( R=0.86, P=0.059). The CYP1A2 activity had a negative correlation with PhIP serum levels at 1 h ( R=0.94, P=0.06) and a positive correlation with urine N-OH-PhIP levels at 0–4 h ( R=0.85, P=0.15). This low level radioisotope method of determining the influence of phenotype on metabolism will significantly improve our understanding of the interrelationships of these pathways and provide a critical foundation for the development of individual risk assessment.

Sulfation of minoxidil by multiple human cytosolic sulfotransferases

Minoxidil is an antihypertensive agent and hair growth promoter that is metabolized by sulfation to the active compound, minoxidil sulfate. Thermostable phenol sulfotransferase (TS PST or P-PST) was initially thought to catalyze the reaction, and the enzyme was designated minoxidil sulfotransferase (MNX-ST). Information about human ST activities toward minoxidil would be useful in developing the capacity to predict individual responses to minoxidil based on tissue levels of STs. Therefore, human STs were studied from platelet homogenates, partially purified platelets, scalp skin high speed supernatants and COS-1 cell cDNA expressed preparations using a radiochemical enzymatic assay with minoxidil as the substrate. Studies showed the presence of TS PST, TL (thermolabile) PST and MNX-ST activities in human scalp skin. Biochemical properties and correlation studies suggested that in addition to TS PST, the TL PST activity, another ST activity or both were involved in the reaction. Partially purified human platelet TL PST tested with minoxidil and dopamine showed identical thermal stabilities and similar responses to the inhibitors 2,6-dichloro-4-nitrophenol (DCNP) and NaCl. To characterize the activity of TL PST toward minoxidil, several biochemical properties of the enzyme expressed from a human liver cDNA clone were investigated. When assayed with minoxidil and dopamine, thermal stabilities of the expressed enzyme were identical and IC 50 values for the inhibitors DCNP and NaCl were similar. It was also demonstrated that cDNA encoded human liver dehydroepiandrosterone sulfotransferase and estrogen sulfotransferase contributed to the sulfation of minoxidil. The results confirm that at least four human STs contribute to minoxidil sulfation. MNX-ST activity represents a combination of ST activities. The data indicate that multiple ST activities should be taken into account in attempts to predict the regulation of minoxidil sulfation and individual responses to minoxidil.

Human Phenol SulfotransferaseSTP2Gene: Molecular Cloning, Structural Characterization, and Chromosomal Localization

Sulfonation is an important pathway in the biotransformation of many drugs, xenobiotics, neurotransmitters, and steroid hormones. The thermostable (TS) form of phenol sulfotransferase (PST) preferentially catalyzes the sulfonation of “simple” planar phenols, and levels of activity of TS PST in human tissues are controlled by inheritance. Two different human liver TS PST cDNAs have been cloned that encode proteins with amino acid sequences that are 96% identical. We have determined the structure and chromosomal localization of the gene for one of these two cDNAs,STP2,as a step toward understanding molecular genetic mechanisms involved in the regulation of this enzyme activity in humans.STP2spans approximately 5.1 kb and contains nine exons that range in length from 74 to 347 bp. The locations of mostSTP2exon–intron splice junctions are identical to those of a gene for the thermolabile form of PST in humans,STM;a rat PST gene; a human estrogen ST (EST) gene,STE;and a guinea pig EST gene. The two initialSTP2exons, IA and IB, were identified by performing 5′-rapid amplification of cDNA ends with human liver cDNA as template. Exons IA and IB are noncoding and represent two different human liver TS PST cDNA 5′-untranslated region sequences. The two apparent 5′-flanking regions of theSTP2gene, regions flanking exons IA and IB, contain no canonical TATA boxes, but do contain CCAAT elements.STP2was localized to human chromosome 16 by performing the PCR with DNA from NIGMS human/rodent somatic cell hybrids as template. Structural characterization ofSTP2will make it possible to begin to study molecular genetic mechanisms involved in the regulation of TS PST activity in human tissue.

Metabolic activation of the N-hydroxy derivative of the carcinogen 4-aminobiphenyl by human tissue sulfotransferases.

The role of human sulfotransferase(s) in the bioactivation of the N-hydroxy (N-OH) metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP) was investigated in vitro with human tissue cytosols. Using an enzymatic assay consisting of a PAPS-regenerating system, [3H]N-OH-ABP, calf thymus DNA and tissue cytosols, the sulfotransferase-mediated metabolic activation of N-OH-ABP was determined as the PAPS-dependent covalent binding of the N-OH substrate to DNA. With cytosols prepared from various tissues, we found that the sulfotransferase(s) in human liver, and to a lesser extent colon, can readily metabolize N-OH-ABP. No PAPS-dependent metabolic activation was detected with cytosols prepared from human pancreas or from the carcinogen target tissue, the urinary bladder epithelium. The N-OH-ABP sulfotransferase activities of liver and colon cytosols from different individuals were highly correlated with their thermostable phenol sulfotransferase (TS-PST) activity (liver, r = 0.99, P < 0.01; colon, r = 0.88, P < 0.01), but not with activities for the thermolabile phenol sulfotransferase (TL-PST; liver, r = 0.29; colon, r = 0.53), or for the dehydroepiandrosterone sulfotransferase (DHEA-ST; liver, r = 0.32; colon, negligible activity). N-OH-ABP sulfotransferase activity was highly sensitive to inhibition by a selective TS-PST inhibitor, 2,6-dichloro-4-nitrophenol (IC50 = 0.7 microM), and by p-nitrophenol, but was unaffected by competitive inhibitors of TL-PST (dopamine) or DHEA-ST (DHEA, DHEA-sulfate). The N-OH-ABP sulfotransferase activity also exhibited thermostability properties similar to that of the TS-PST. From these data, we conclude that human liver TS-PST but not TL-PST or DHEA-ST can metabolically activate the proximate human carcinogen N-OH-ABP to a reactive sulfuric acid ester intermediate that binds covalently to DNA. In addition, in view of the putative role of N-OH-ABP as a major transport form of the carcinogen to the urinary bladder and of the absence of sulfotransferase activity in this tissue, we hypothesize that sulfotransferase activation in the liver may actually decrease the bioavailability of N-OH-ABP toward extrahepatic tissues and thus serve as an important overall detoxification mechanism for the urinary bladder.

Human platelet thermostable phenol sulfotransferase: Assay of frozen samples and correlation between frozen and fresh activities

Sulfate conjugation catalyzed by phenol sulfotransferase (PST, EC 2.8.2.1) is an important metabolic pathway for catecholamines, endogenous phenolic compounds and catechol drugs [l--4]. Thermolabile (TL) and thermostable (TS) forms of PST are present in human tissues [S-6]. The platelet is an easily obtained tissue that is useful for the study of human PST regulation [3-4,7-lo]. Only TS PST in fresh platelets prepared and assayed immediately differs between American blacks and whites in levels of activity and thermal stability [ll-121. The ability to collect, prepare and freeze multiple samples prior to assay would simplify family studies of platelet PST from American blacks. We had three objectives in this project. First, we modified the method of platelet preparation to allow frozen storage of samples and we investigated biochemical properties of the frozen enzyme. Second, we compared levels of TS PST activities and thermal stabilities from frozen and fresh samples. Third, we used the modified assay to measure TS PST activity and thermal stability in platelet samples from randomly selected subjects. The biochemical properties and activities of TS PST measured with p-nitrophenol by the modified assay compared well with the results obtained with fresh samples. The levels of TS PST activity and distributions of thermal stabilities detected with the frozen samples were essentially identical to results we reported with fresh samples [12].

Sulfation pharmacogenetics: correlation of human platelet and small intestinal phenol sulfotransferase.

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic drugs. All human tissues studied contain a thermostable (TS) form of PST, which catalyzes the sulfate conjugation of "simple" phenols such as p-nitrophenol, and a thermolabile (TL) form, which catalyzes the sulfation of dopamine and other monoamines. In the present study we tested the hypothesis that genetically controlled levels of TS and TL PST activity in the platelet, as well as inherited variations in the thermal stability of platelet TS PST, might reflect those same characteristics of the enzyme in the less accessible tissue, human small intestinal mucosa. Platelet TS and TL PST activities and TS PST thermal stability were measured in blood samples from 45 randomly selected healthy subjects, and 14 of those subjects were selected to have intestinal biopsies performed. There was a significant correlation between levels of platelet and jejunal mucosal TS PST activity (rs = 0.574, p less than 0.030), but there was not a significant correlation between levels of TL PST activity in the two tissues (rs = 0.265, p = 0.368). There was also a significant correlation between the trait of TS PST thermal stability in the two tissues (rs = 0.828, p less than 0.0001). These observations suggest that inherited variations in TS PST activity and thermal stability in an easily obtained tissue, the platelet, might be used to predict individual differences of those properties of the enzyme in the human small intestine, an organ that plays an important role in drug metabolism.

Genetic polymorphism for human platelet thermostable phenol sulfotransferase (TS PST) activity.

Platelet TS PST basal activity and thermal stability were measured in blood samples from 237 individuals in 50 nuclear families. Significant correlations were found among first degree relatives, confirming the previously reported familial aggregation of TS PST basal activity and thermal stability. Commingling analysis of basal TS PST activity provided evidence for multiple component distributions, and after transformation to remove skewness, segregation analysis supported a major gene hypothesis. For TS PST thermal stability, commingling analysis also provided evidence for multiple component distributions. However, segregation analyses were equivocal with regard to the presence of a major gene for thermal stability, since support for a major gene model depended on skewness. Bivariate commingling analysis, which examined thermal stability by simultaneously considering basal activity and activity after heating, suggested that genotypes, as defined by the inferred component distributions for TS PST activity, differ in thermal stability. A three-allele model is proposed as one hypothesis that may account for the combined results of basal activity and thermal stability. The results of this study indicate that a major gene polymorphism in conjunction with polygenic inheritance plays an important role in the regulation of both level of activity and thermal stability of this important drug-metabolizing enzyme in humans.

Triiodothyronine: A Substrate for the Thermostable and Thermolabile Forms of Human Phenol Sulfotransferase*

The enzyme(s) responsible for the sulfate conjugation of L-T3 in man has not been characterized. T3 sulfotransferase (T3-ST) activity was characterized in normal human liver tissue obtained during clinically indicated surgical resection. Subcellular distribution studies showed that the T3-ST activity was localized to the cytoplasmic fraction. This finding raised the possibility that T3-ST activity might be similar to the 2 previously identified forms of cytoplasmic phenol sulfotransferase (PST) found in human tissue. A thermostable (TS) form of PST catalyzes the sulfate conjugation of micromolar concentrations of p-nitrophenol, and a thermolabile (TL) form catalyzes the sulfate conjugation of dopamine and other monoamines. Thermal stability and enzyme inhibitor experiments showed that T3-ST activity in pooled liver homogenates was very similar to the TS form of PST. The apparent similarity of T3-ST to TS PST was studied further by measuring T3-ST, TS PST, and TL PST activities in 20 individual liver samples. T3-ST activities correlated significantly with TS PST activities (r = 0.939; P less than 0.001) measured with p-nitrophenol, but not with TL PST activities (r = -0.118; P greater than 0.6) measured with dopamine. However, sulfation of T3 by the TL form of the enzyme might have been masked by the 18-fold higher specific activity of TS than TL PST in human liver homogenates. When the two forms of PST were separated by ion exchange chromatography, T3 was found to be a substrate for both the TS and TL forms of PST. "True" Km values for T3 were similar for TS and TL PST (81 and 127 microM, respectively).

Phenol sulfotransferase inheritance.

1. Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of many phenolic and catechol neurotransmitters. Human tissues contain both thermostable (TS) and thermolabile (TL) forms of PST that differ in their substrate specificities, inhibitor sensitivities, physical properties, and regulation. 2. Individual variations in the levels of activity of both TS and TL PST in the human platelet are strongly influenced by inheritance. 3. Individual differences in the level of platelet TS PST activity are correlated with individual variations in the activity of this form of the enzyme in human cerebral cortex, liver, and intestinal mucosa. 4. There are also individual familial differences in the thermal stability of TS PST in the platelet. These differences are correlated with individual variations in the thermal stability of TS PST in cerebral cortex, liver, and intestinal mucosa. 5. Individual variations in the thermal stability of TS PST in hepatic tissue are associated with the presence of one or both of a pair of TS PST isozymes that can be separated by ion-exchange chromatography and that differ in their thermal stabilities. 6. This series of observations suggests that a structural gene polymorphism may be one mechanism by which inheritance controls TS PST in humans. The isozymes of TS PST in liver may represent the products of alternative alleles for this polymorphism, alleles that might control the structure of TS PST in many human tissues.

Human liver phenol sulfotransferase: Assay conditions, biochemical properties and partial purification of isozymes of the thermostable form

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic and catechol drugs and neurotransmitiers. Human platelets and brain contain at least two forms of PST. One form is relatively thermolabile (TL) and catalyzes the sulfate conjugation of monoamines such as dopamine. The other is thermostable (TS) and catalyzes the sulfation of “simple” phenols such as phenol and p-nitrophenol. We found that homogenates of human liver also contain two forms of PST that are similar to brain and platelet TL and TS PST with regard to substrate specificities, thermal stabilities and sensitivities to inhibitors. Optimal conditions were determined for the assay of these two activities in human liver homogenates. The apparent K m of liver homogenate TL PST for dopamine was 27 μM. The apparent K m of the TS form of the enzyme for p-nitrophenol was 0.94 μM. Human liver TS PST also catalyzed the sulfate conjugation of dopamine, but with an apparent K m of 5 mM, over two orders of magnitude higher than that of TL PST. Two different peaks of TS PST activity were separated from the TL activity by ion exchange chromatography of human liver preparations. Both peaks of TS PST activity were partially purified and characterized. Both had similar substrate specificities and inhibitor sensitivities. K m values of TS PST peak I for p-nitrophenol and for 3'-phosphoadenosine-5'-phosphosulfate were 0.91 and 0.86 μM, respectively, while the K m values of TS PST peak II for these two cosubstrates for the reaction were 0.43 and 0.64 μM, respectively. However, the TS PST activity in peak II was significantly more thermolabile than was the activity in peak I. These results are compatible with the conclusion that human liver homogenates contain at least two forms of PST, forms with properties similar to those of TS and TL PST in homogenates of human cerebral cortex and platelets. In addition, human liver contains two isozymes of TS PST.

Human pituitary phenol sulfotransferase: biochemical properties and activities of the thermostable and thermolabile forms.

Pituitary tissue contains phenol sulfotransferase (PST), the enzyme that catalyzes the sulfate conjugation of monoamine neurotransmitters. We carried out these studies with pituitaries obtained 21.3 +/- 3.0 h postmortem (mean +/- SEM; n = 21) to determine whether the biochemical properties and variations in levels of human pituitary PST activities were similar to those of PST in platelets from control subjects. PST in the human platelet has been studied thoroughly because of the possibility that platelet PST might reflect levels of PST activity in other tissues such as the pituitary and brain. Our results demonstrated 2 forms of the pituitary enzyme that were similar to the thermostable (TS) and thermolabile (TL) forms of platelet PST with regard to assay conditions, pH optima, Km values for multiple substrates, responses to 2,6-dichloro-4-nitrophenol (DCNP), and thermal stability properties. Pituitary samples also were obtained at autopsy 6.3 +/- 0.33 h (mean +/- SEM; n = 3) after death to determine the effects of storage at 4 degrees C on PST activities. After storage for 6-18 h, 83-99.6% of the TS PST activity remained and 44-66.9% of the TL PST activity remained. Pituitary TS PST activity in samples obtained within 12.1 +/- 3.25 h after death was 121.0 +/- 49.1 units/mg protein (mean +/- SEM; n = 7) with a range from 9.7 to 367.6. TL PST activity was 35.6 +/- 11.6 units/mg protein (mean +/- SEM; n = 6) with a range from 6.1 to 80.7. Wide variations of both enzyme activities were also present in 3 pituitary tumor samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermolabile and thermostable human platelet phenol sulfotransferase. Substrate specificity and physical separation.

Human platelets contain at least two forms of phenol sulfotransferase (PST), a thermolabile (TL) form for which dopamine is a substrate and a thermostable (TS) form for which micromolar concentrations of phenol can serve as substrate. At higher concentrations phenol is also a substrate for the TL form. Studies of the regulation and the possible clinical value of measurements of platelet PST have been hampered because there is no specific substrate for the TS form of the enzyme. The purposes of these experiments were to determine whether there might be a better substrate than phenol for use in measurement of the activity of the TS form of platelet PST, and to attempt to physically separate the two forms of the platelet enzyme. The results of substrate kinetic, thermal stability, and inhibitor studies performed with platelet homogenates were all compatible with the conclusion that p-nitrophenol and 6-OH-melatonin were substrates for both the TS and TL forms of platelet PST. Norepinephrine, epinephrine and 5-OH-tryptamine were substrates for only the TL form. The apparent Km constants of the two forms of PST for p-nitrophenol differed by 7,100-fold when measured in platelet homogenates. This difference was 200 times greater than that which has been reported for phenol. Therefore, p-nitrophenol is the preferred substrate for measurement of the TS PST activity if interference by the TL activity is to be avoided. This information made it possible to use p-nitrophenol as a substrate in experiments designed to separate the two forms of platelet PST.(ABSTRACT TRUNCATED AT 250 WORDS)