1 MODULATION OF STEROID SULFONATION BY SMALL MOLECULES THAT INTERACT WITH SULFOTRANSFERASES AND SULFAT A SES By SRIRAM AMBADAPADI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFIL LMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012
2 2012 Sriram Ambadapadi
3 To my parents
4 ACKNOWLEDGMENTS This dissertation would not be possible without enormous conceptual and procedural help I r eceived from my advisor and the chair of my degree supervisory committee Prof. Margaret James. I thank her for the equanimity with which she lent her time and patience to my academic endeavors good, bad and ugly. My sincere thanks should also go to the members of my degree supervisory committee Prof. Kenneth Sloan, Prof Hendrik Luesch and Prof. Ben Dunn who gave their time and attention readily when I requested it. Also, I thank Dr. Sergiu P. Palii for his help in mass spectrometric analysis of sulfotra nsferase assays it was if not for all the help I got from our biological scient ist/lab manager Laura Faux. I express my D e partment of Medicinal Chemistry. Special thanks should also go to the undergraduates I worked with Peter Bo u los and Peter Emile. Finally, I should thank my family and friends without whose support, I would neither be able to undertake nor finish this work
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 INTRODUCTION TO SULFONATION OF XENOBIOTICS ................................ ..... 15 Sulfotransf erases ................................ ................................ ................................ .... 15 Structure and Mechanism ................................ ................................ ................. 18 Inhibition of Catalysis ................................ ................................ ....................... 22 Sulfatases ................................ ................................ ................................ ............... 22 Estrogens and Breast Cancer ................................ ................................ ................. 25 Product Switching and Sti mulation of Overall Catalysis by Celecoxib .................... 28 Hypotheses and Specific Aims ................................ ................................ ............... 29 Hypothesis 1 ................................ ................................ ................................ ..... 29 Specific aim 1 ................................ ................................ ............................. 29 Specific aim 2 ................................ ................................ ............................. 29 Specific aim 3 ................................ ................................ ............................. 30 Hypothe sis 2 ................................ ................................ ................................ ..... 30 Hypothesis 3 ................................ ................................ ................................ ..... 30 2 CELECOXIB EFFECT ON ESTRADIOL ANALOGUE SU LFONATION ................. 38 Introduction ................................ ................................ ................................ ............. 38 Materials and Methods ................................ ................................ ............................ 40 Bacterial Expression of SULT2A1 ................................ ................................ .... 40 SULT2A1 Assay ................................ ................................ ............................... 41 LC MS/MS An alysis ................................ ................................ ......................... 41 Effect of Celecoxib on Sulfatase Activity ................................ .......................... 42 Results ................................ ................................ ................................ .................... 43 Estradiol Analogue Sulfonation in the Presence of Celecoxib .......................... 43 Effect of Celecoxib on Sulfatase Activity ................................ .......................... 46 Conclusions and Discussion ................................ ................................ ................... 46 3 SPECIES DIFFERENCES IN THE CELECOXIB EFFECT ................................ ..... 55 Introduction ................................ ................................ ................................ ............. 55
6 Materials and Methods ................................ ................................ ............................ 55 Assay of Rat and Sheep Liver Cytosol ................................ ............................. 55 HPLC Analysis ................................ ................................ ................................ 56 Results ................................ ................................ ................................ .................... 56 Sheep and Male Rat ................................ ................................ ......................... 56 Female Rats ................................ ................................ ................................ ..... 57 Discussion ................................ ................................ ................................ .............. 57 4 LIGAND DOCKING STUDIES ON THE CELECOXIB MODULATION OF ESTRADIOL ANALOGUE SULFONATION ................................ ............................ 65 Introduction ................................ ................................ ................................ ............. 65 Method ................................ ................................ ................................ .................... 66 Results ................................ ................................ ................................ .................... 68 Dockings in the Absence of Celecoxib ................................ ............................. 69 Dockings in the Presence of Celecoxib ................................ ............................ 72 Rat vs. Human S ULT ................................ ................................ ....................... 74 Discussion ................................ ................................ ................................ .............. 75 5 TRICLOSAN AS THE SUBSTRATE AND INHIBITOR OF CYTOSOLIC SULFOTRANSFERASES ................................ ................................ ....................... 93 Introduction ................................ ................................ ................................ ............. 93 Materials and Methods ................................ ................................ ............................ 96 Expression of Recombinant Sulfotransferases ................................ ................. 96 Triclosan as a Substrate ................................ ................................ ................... 97 Effect of Triclosan on Estradiol Sulfonation in the Presence of Celecoxib ....... 99 Triclosan Inhibition of Sheep Placental Sulfatase Activity ................................ 99 Results ................................ ................................ ................................ .................. 100 Triclosan as a Substrate ................................ ................................ ................. 100 Triclosan Inhibition of Sulfotransferase Activities ................................ ........... 102 Effect of Triclosan on Estradiol Sulfonation in the Presence of Celecoxib ..... 102 Triclosan Inhibition of Sheep Placental Sulfatase Activity .............................. 103 Discussion ................................ ................................ ................................ ............ 103 6 CONCLUSIONS ................................ ................................ ................................ ... 126 LIST OF REFERENCES ................................ ................................ ............................. 132 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 147
7 LIST OF TABLES Table page 3 1 Ef ..................... 62 3 2 ............................ 63 3 3 .......................... 64 4 1 Activities (pmol/min/mg protein) of the substrates in the absence of celecoxib .. 92 5 1 Triclosan sulfonation by major human cytosolic sulfotransferases ................... 124 5 2 The IC 50 ( M) values for triclosan inhibition of 4 methylumbelliferysulfate hydrolysis ................................ ................................ ................................ ......... 125
8 LIST OF FIGURES Figure page 1 1 Depiction of constituent parts of the name of SULT enzyme .............................. 31 1 2 The expression values for each human sufotransferase enzymes in four different tissues: liver, sm all intestine, kidney, and lung ................................ ..... 3 2 1 3 SULT2A1 structure shown with docked D HEA and PAP ................................ .... 33 1 4 A proposed sulfonation mechanism by SULTs ................................ ................... 34 1 5 Crystal structure of STS. PDB ID 1P49, reported in (Hernande z Guzman et al., 2003). ................................ ................................ ................................ ........... 35 1 6 Catalytic mechanism for steroid sulfatase ................................ .......................... 36 1 7 Estradiol generation in tissues from the transpo rt forms of steroids ................... 37 2 1 Structures of estradiol analogues tested for the effect of celecoxib modulation 50 2 2 Effect of cel estradiol by SULT2A1 enzyme .......... 51 2 3 estradiol and 3 Methoxyestradiol sulfonation by SULT2A1 enzyme ................................ ........... 52 2 4 estradiol analogues by SULT2A1 enzyme ................................ ................................ ........ 53 2 5 Effect of celecoxib on sulfon ation of 400 nM catechol estrogen sulfonation by SULT2A1 enzyme ................................ ................................ .............................. 54 3 1 Representative chromatograms of sulfonation of 50 nM 17 E2 in the presence and absence of celecoxib ................................ ................................ ... 60 3 2 Representative chromatograms of sulfonation of 50 nM estradiol by female rat liver cytosol ................................ ................................ ................................ .... 61 4 1 E2 (magenta ) docked in SULT2A1 .............. 79 4 2 Epi T (blue), T (green) docked in SULT2A1 ................................ ....................... 80 4 3 6D E2 (magenta), 9D E2 (gold), Eqn (purple) and Eq ( green) docked in SULT2A1 ................................ ................................ ................................ ............ 81 4 4 4OH E2 (cyan), 2OH E2 (light green) docked in SULT2A1 ................................ 82 4 5 E2 (green) and 3Me E2 (salmon) docked in SULT2A1 ............ 83
9 4 6 E2 and celecoxib docked in SULT2A1 ................................ ........................ 84 4 7 E2 (d ark blue), AD (light blue) and DHEA (green) docked in SULT2A1 in the presence of celecoxib ................................ ................................ ................... 85 4 8 Epi T (blue), T (green) docked in SULT2A1 in the presence of celecoxib. ......... 86 4 9 6D E2 (magenta), 9D E2 (gold), Eqn (purple) and Eq (green) docked in SULT2A1 in the presence of celecoxib ................................ ............................... 87 4 10 4OH E2 (cyan), 2OH E2 (light green) docked in SULT2A1 in the presence of celecoxib ................................ ................................ ................................ ............ 88 4 11 E2 (green), 3Me_E2 (purple) and Eq (salmon) docked in SULT2A1 in the presence of celecoxib ................................ ............................... 89 4 12 Comparison between human SULT2A1 and rat ST 60 ................................ ...... 90 4 13 An overlap showing the binding of PAP or PAPS molecules in crystal structures availabl e in the Protein Data Bank ................................ ..................... 91 5 1 Structures of triclosan and thyroid hormones, triiodothyronine and thyroxine. 108 5 2 Represen tative thin layered chromatogram of SULT1A1 sulfonation of triclosan ................................ ................................ ................................ ............ 109 5 3 Sulfonation of increasing triclosan (0 to 1000 M) by SULT1A1 ....................... 110 5 4 Sulfonation of increasing triclosan (0 to 1000 M) by SULT1B1 ....................... 111 5 5 Sulfonation of increasing triclosan (0 to 2000 M) by SULT1E1 ....................... 112 5 6 Sulfonation of increasing triclosan (0 to 2000 M) by SULT1A3 ....................... 113 5 7 Sulfonation of increasing triclosan (0 to 1000 M) by SULT2A1 ....................... 114 5 8 The mechanism of substrate inhibition observed with SULT1A1 and SULT1B1 with triclosan as the substrate ................................ .......................... 115 5 9 Triclosan inhibition SULT1A1 activity with 4 M [ 14 C] p nitrophenol (p Np) as the substrate. ................................ ................................ ................................ .... 116 5 10 Triclosan inhibition SULT1B1 activity with 100 M triiodothyronine (T3) as the substrate ................................ ................................ ................................ ........... 117 5 11 Triclosan inhibition SULT1A3 activity with 10 M dopamine as the substrate .. 118 5 12 Autoradiogram the reaction mixtures measuring triclosan inhibition of dop amine sulfonation ................................ ................................ ....................... 119
10 5 13 Autoradiogram the reaction mixtures measuring triclosan inhibition of T3 sulfonation ................................ ................................ ................................ ........ 120 5 14 Triclos an inhibition of E2 3S generation b y pooled human liver cytosol ......... 121 5 15 Triclosan inhibition of 4 Methylumbelliferyl sulfate hydrolysis by sulfatase enzyme. ................................ ................................ ................................ ............ 122 5 16 Triclosan inhibition of estrone sulfate hydrolysis by steroid sulfatase in sheep placental microsomes ................................ ................................ ....................... 123
11 LIST OF ABBREVIATION S 17 E2 17 Estradiol 17 E2 17 Estradiol HSD 17 Hydroxysteroid dehydrogenase 2 OHE2 2 Hydroxyestradiol 3Me E2 3 Methoxyestradiol 4 OHE2 4 Hydroxyestradiol 6D E2 6 Dehydroestradiol 9D E2 9 Dehydroestradiol AD Androstenediol ARS Arylsulfatase BSA Bovine serum albumin CE Catechol estrogen COX 2 Cyclooxyg enase 2 DEAE Diethylaminoethyl DHEA Dehydroepiandrosterone DMSO Dimethyl sulfoxide DTT Dithiothreitol E1 Estrone EDTA Ethylenediaminetetraacetic acid EE Ethynyl estradiol EPA Environmental Protection Agency Epi T Epitestosterone Eq 17 Dihydroequilin Eqn 1 7 Dihydroequilenin
12 EST Estrogen sulfotransferase FDA Food and Drug Administration FG Formylglycine H ESI Heated electrospray ionization HFG Hydroxyformylglycine HPLC High pressure liquid chromatography IPTG Iso pr o pyl beta D thiogalactopyranoside MSD Mult iple sulfatase deficiency NCBI National Center for Biotechnology Information PAP Phosphoadenosine Phosphate PAPS Phos phoadenosine Phosphosulfate PDB Protein data bank Pic A Tetrabutylammonium sulfate p NP p n itrophenol PSB loop Phosphate bindin g loop PST Phenol sulfotransferase SDS PAGE S odium dodecyl sulfate polyacrylamide gel electrophoresis STS Steroid sulfatase SULT Sulfotransferase T Testosterone T3 T riiodothyronine TEA Triethanolamine Tris Tris(hydroxymethyl)aminomethane UDP Uridine diphosphate glucose
13 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MODULATION OF STEROID SULFONATION BY SMALL MOLECULES THAT INTERACT WITH SULFOTRANSFERASES AND SULFAT A SES By Sriram Ambadapadi December 2012 Chair: Margaret James Major: Pharmaceutical Sciences Sulfonation a major ph ase II conjugation reaction turns o therwise highly lipophilic compounds water soluble and ma kes them amenable to excretion via the kidneys. Sulfonated forms of compounds such as 17 Estradiol (17 E2) also act as transport forms which are made available to various tissues by the plasma. Several small molecules interact with sulfotransferases to alter their activity. In this study, interaction of two compounds, celecoxib and triclosa n, were explored at the molecular level. It was observed previously that 17 E2 which upon sulfotransferase2A1 (SULT2A1) catalysis forms 17 E2 3 sulfate as the major product and also forms 17 E2 17 sulfate as the minor product, switches its sulfonation pattern in the presence of the cox 2 inhibitor, celecoxib to form more 17 E2 17 sulfate in human liver cytosol T o investigate the reason for this switching at the molecular level steroids analogous to 17 E2 were selected for the study of their sulfonat ion by SULT2A1 in the presence of celecoxib. Enzyme kinetic data when coupled with in silico ligand docking studies suggest s that celecoxib binds in the large binding site of SULT2A1 and while prohibiting the normal binding of the substrates facilitates for appropriately shaped substrates a binding mode that allows more 17 sulfonation.
14 The effect of celecoxib on the sheep and rat liver cytosols was also tested in vitro with 17 E2 to see if they replicate the human model. There was inhibition of sulfonation but no switching in female sheep and male rat. In female rat there was no observable effect of celecoxib, but it was interesting to note that 17 E2 17 sulfate was the major product of 17 E2 sulfonation. Triclosan, an antibacterial used in preparations such soaps and tooth pastes has been shown to interact with off target proteins Since it is shown to be excreted mainly as its conjugates, studies were undertaken to identi fy the major isoforms of sulfotransferase enzymes bringing about this biotransformation. SULT1 B 1 and SULT 1A 1 with V max /K m values of 820 and 320 ml/min/mg are the most active isoforms. Triclosan also was inhibitory towards the activities of other major huma n sulfotransferase and sulfa tase enzymes.
15 CHAPTER 1 INTRODUCTION TO SULFONATION OF XENOB IOTICS Sulfotransferases S ulfonation in biomolecules was found as early as 1876 (Baumann, 1876) Isolation of the universal sulfonate donor phosphoadenos ine phosphosulfate (PAPS) was completed by 1956 (Robbins and Lipmann, 1956) Sulfonation is the transfer of sulfuryl group from PAPS to a nucleophilic substrate and is catalyzed by a family of enzymes called sulfotransferases (SULTs). Two different types of SULTs, classified depending on their cellular localization, are cytosolic and membrane bound SULTs. Membrane bound SULTs sulfonate large biomolecules such as carbohydrates an d proteins. Cytosolic SULTs sulfonate endogenous compounds such as steroid and thyroid hormones, bioamines and also numerous xenobiotics including drugs and environmental pollutants. Detoxification of xenobiotics and endogenous molecules by SULTs is based on the sulfonation of those relatively hydrophobic molecules into sulfuric acid esters which can be excreted in urine readily owing to their water solubility and recognition by transport proteins. Phenolic and alcoholic drugs and endogenous compounds such as steroid hormones, neurotransmitters and bile acids upon sulfonation form stable sulfate esters that are usually not biologically active. There are however some examples of drugs like minoxidil (Meisheri et al., 19 88; Meisheri et al., 1993) and cicletanine (Garay et al., 1995) whose sulfates are more active than the drugs themselves. Some benzylic and allylic alcohols, on the other hand, were shown to be activated by sulfona tion to form electrophilic mutagens which can induce tumors (Miller, 1994; Glatt et al., 1995)
16 SULTs along with cytochrome P450s and UDP glucuronosyltransferases are very important metabolic enzymes in drug biotrans formation. Of this group, SULTs are expressed even in the human fetus (Barker et al., 1994; Hume and Coughtrie, 1994; Hume et al., 1996; Richard et al., 2001; Stanley et al., 2001) and form their only chemical defen se as many other xenobiotic metabolizing enzymes are not expressed significantly until after the birth (Coughtrie et al., 1988; Hakkola et al., 1998) Different SULTs catalyzing the sulfonation of a variety of endo and xenobiotics have been known for a long time and initially the names given for these enzymes were indicative of their substrate preferences. This arbitrary naming is both inconvenient for broader usage among researche r s and impractical, as there are su bstrates that can be sulfonated by more than one SULT. F or example, estradiol is a high affinity substrate of the enzyme estrogen sulfotransferase (previously known as EST) but can also be sulfonated by what is referred to as phenol sulfotransferase (previ ously known as PST) in large capacity. A system of nomenclature was proposed (Blanchard et al., 2004) that placed all the cytosolic SULTs in a single superfamily which is divided into families and subfamilies of enzymes depending on the similarity of their amino acid sequences. To be members of the same family the enzymes should share at least 45% of the amino acid sequence and at least be 60% id entical to be in the same subfamily. Figure 1 1 depicts the construction of a name of an enzyme according to the new nomenclature along with its constituent parts. The 13 known human cytosoli c SULTs have been classified in to four families, SULT1, SULT2, S ULT4 and SULT6. The SULT1 family is by far the largest with four
17 subfamilies (1A, 1B, 1C and 1E) and nine members viz. 1A1, 1A2, 1A3, 1A4, 1B1, 1C1, 1C2, 1C3, and 1E1. Two known genes encode for three SULT2 family members SULT2A1, 2B1a and 2B1b. The remai ning two human SULTs are SULT4A1 and SULT6B1. The similarity in amino acid sequences of the individual enzymes among SULT families does not always translate into similar substrate specificities. It is the substrate binding pocket and the similarity of the crucial amino acids in this pocket that example share about 93% overall similarity, but the latter has a K m value about 3600 times tha n that of the former towards sulfonation o f p nitrophenol, a preferred substrate of SULT1A1. Similarly, for sulfonation of dopamine, an endogenous substrate of SULT1A3, the K m value of SULT1A1 is about 35 times greater compared to SULT1A3 (Veronese et al., 1994) Nevertheless, sulfonation of some general classes of substrates can be attributed to specific S ULT s, keeping in mind all the time that the se substrate specif icities overlap more as a rule than as an exception. Among the two main families SULT1 is designated as phenol sulfotransferase family and SULT2 as the hydroxysteroid family based on their preference for aromatic and aliphatic hydroxy groups respectively. SULT1A1, the most abundant form in the liver has very broad substrate selectivity and is the main xenobiotic metabolizing SULT. It can sulfonate phenols, naphthols, benzylic alcohols, arom atic amines and hydroxylamines (Glatt and Meinl, 2004) SULT1A3 sulfonates catecholamines with high selectivity and is expressed only in primates (Dajani et al., 1998; Eisenhofer et al., 1999) SULT1B1 sulfonates thyroid hormones and small phenolic compounds (Fujita et al., 1997) whereas SULT1C1 acts
18 on aryl hydroxlamines and iodothyronines (Li et al., 2000) SULT1E1 is also called estrogen sulfotransferase and has greater affinity for estrogens and th ough it sulfonates other phenoli c compounds it is one of the most selective SULTs a s evidenced by its preference for estrogens (Falany et al., 1995) SULT2A1 is the main hydroxysteroid s ulfotransferase and its main substrates are dehydroepiandrosterone, androgens, bile acids and estrogens (Comer et al., 1993) The substrates for SULT4A1 and SULT6B1 have not been identified to date. SULT4A1 is highly conserved and is expressed in brain (Falany et al., 2000) and SULT6B1 gene is expressed in testis of primates (Freimuth et al., 2004) There have been very few studies determining the quantitative rela tionships between substrate structure and catalytic activity of SULTs. One of the studies determined the optimal length of alkyl phenols for sulfonation by SULT1A1 and SULT1A2 to be between 1 4 carbon atoms. Also the substitution of the alkyl chain at 2 po sitio n seemed to be best for the cat alytic efficiency (Harris et al., 2000) Similarly for SULT2A1 it was found that benzyl alcohols para substituted w ith n pent y l alkyl groups showed the best catalytic efficiency. For n alcohols the chain length of 9 11 carbon atoms was optimal (Chen et al., 1996) Distribution of various SULTs in human tissues of major importance in biotransformation is depicted in Figure 1 2. Ex pression levels were determined by quantitative immunoblotting in liver, small intestine, kidney and lung (Riches et al., 2009) Structure a nd M echanism Sulfotransferases are globular molecules, as evidenced by the accumulated crystallographic evidence, and usually exist as homo or heterodimers (Kiehlbauch et
19 al., 1995) The role of dimerization in the function of these enzymes is yet to be understood. They all share a simple basic fold in which a four or five sheet is sheet contains two distinct regions for binding of PAPS and the substrate and the loops around it are more ordere d upon binding of either the substrate or the universal co factor, PAPS (Allali Hassani et al., 2007) PAPS binding region is highly conserved at the amino acid level and the substrate binding region is where the di fferences in the individual SULTs manifest. SULTs resemble nucleotide kinases in their connectivity and secondary structures. The super imposable structural features between these two enzyme groups when coupled with the similarity in core structures of phos phate and sulfonate groups suggest a possible co evolution of the two enzyme systems (Negishi et al., 2001) Figure 1 3 depicts a representative SULT structure (SULT2A1) along with a substrate and PAPS. Mouse estrogen sulfotransferase was the first of the SULTs to be crystallized and it was reported in a co crystalized form with PAPS and the catalysis substrate estradiol (Kakuta et al., 1997) Subsequently 11 of the human enzymes have been crystallized with various substrates and the co substrate analog PAP. PAP is more convenient for the crystallization processes as it does not ha ve the labile sulfate group of PAPS d oes and at the same time provides useful information about the co factor binding. The majority of the interactions of the PAPS molecule with the enzyme are provided by two structurally and sequentially conserved motifs in the SULT. A strand loop helix mot if comprising the phosphate binding loop (PSB loop) forms hydrogen phosphate of the PAPS molecule and helix 6 of the strand turn helix phosphate group of PAPS. The consensus sequences
20 PKT/GTTW/AL and IT/YV/I/LL phosphate binding regions have been used as a criterion for identifying the newly cloned cDNAs of SULTs (Kakuta et al., 1998b) The substrate binding region is where the individual amino acids play a key role in substrate specificity. For exam ple, Tyr 81 of SULT1E1 provides a steric hindrance to dehydroepiandrosterone (DHEA) binding whereas the Y81L mutation facilitates DHEA sulfonation (Petrotchenko et al., 1999) This deep hydrophobic substrate binding pocket is large enough to accommodate two substrate molecules, one of which is usually is in a non reactive mode. This has been proposed as the reason for frequently observed substrate inhibition at high sub strate concentrations in sulfotransferases such as SULT2A1 (Pedersen et al., 2000; Rehse et al., 2002) The catalytic histidine residue (at His107 in SULT1E1, for example) is vital and when mutated abolishes the ac tivity of the enzyme (Kakuta et al., 1998a) During catalysis it abstracts the pro ton from the hydroxy group of the substrate thereby leaving the oxygen nucleophili c enough to attack the PAPS sulfur. Mutagenesis studies on some membrane bound SULTs provided important information about other active site residues participating in catalysis. Lys59 along with His118 is identified to be crucial for the activity of flavon ol 3 sulfotransferase (Marsolais and Varin, 1995; Marsolais and Varin, 1997) and a conserved residue Ser197 in HNK 1 sulfotransferase was found to be regulating the catalytic activity (Ong et al., 1999) According to a proposed mechanism for sulfonation (shown in Figure 1 4) the side phosphate of the PAPS when the substrate is not present by its hydrogen bonding int eraction with the
21 oxygen on the side chain Ser. When the substrate is bound in the active site of the enzyme His abstracts the proton from the acceptor group leaving the oxygen nucleophilic. The interaction with this nucl eo philic group leads to accumulatio n of to the sulfonate which in turn forces the side chain nitrogen of Lys to interact with itself thereby facilitating the sulfonate dissociation. The Ser residue plays a dual role here of interacti phosphate to position PAPS in catalytic mode and preventing the hydrolysis of PAPS before substrate binding by keeping the Lys side chain nitrogen away from the bridging phosphate (Negishi et al., 2001) A crystal structure of SULT1A3 with no acceptor subst rate or PAPS showed that the Ser was not in position to form a hydrogen bond with the Lys (Bidwell et al., 1999) phosphate of PAPS does the side chain of Ser interact with that of Lys. This interaction may help prevent the dissociation of PAPS prior to substrate binding. Only after the generation of phosphate group does this interaction break and subsequently allows dissociation of the sulfonate. According to this reaction mechanism the catalysis req uires the formation of a ternary complex of the enzyme, along with the substrate and the PAPS for the formation of products. It requires the binding to be sequential wherein the PAPS binds to the enzyme before the acceptor substrate. But the order of the b indings has been found to vary and there has been evidence for both ordered bi bi and random bi bi kinetics (Chap man et al., 2004)
22 Inhibition of C atalysis Certain drugs, environmental and dietary chemicals along with some endogenous compounds can inhibit the SULTs. Role of sulfonation in the detoxification of endogenous molecules like steroid and thyroid hormon es justifies the search for specific inhibitors. Highly potent inhibitors acting on the PAPS binding sites can arrest the family as a whole but are not specific enough to inhibit individual enzymes. Dietary flavonoids (Ghazali and Waring, 1999; De Santi et al., 2002; Vietri et al., 2003) of which quercetin is the most potent and constituents of red wine such as cathechin (Gibb et al., 1987) were found to inhibit SULT 1 family. The hydroxylated metabolites of polychlorinated biphenyls (PCBs) are inhibitors of SULTs metabolizing estrogens and thyroid hormones, mainly SULT 1 and 2 families (Wang et al., 2005; Liu et al., 2006; Wang and James, 200 6) Triclosan, a commonly used antimicrobial product is a potent inhibitor of SULT1E1 (Wang et al., 2004; James et al., 2010) Sulfatases Sulfatases are a family of enzymes that catalyze the hydrolysis of sulfate e sters (CO S) and sulfamates (CN S), thus playing a role of reversing the SULT actions. Sulfatases play a key role in hormone regulation, gamete interactions and in bone development by catalyzing the hydrolysis of steroid and thyro id hormone sulfates as wel l as the sulfates of carbohydrates, proteoglycans and glycolipids. Interest in sulfatases has increased after the discovery that their deficiencies led to a variety of inherited lysosomal disorders (Hanson et al., 2004) Seventeen human sulfatases have been identified (Parenti et al., 1997; Diez Roux and Ballabio, 2005) They are about 500 to 600 amino acids in length when translated and are targeted for secretory pathway by extensive glycosylation (Diez Roux and
23 Ballabio, 2005) Sulfatases can be divided into soluble and membrane bound enzymes based on their cellular localization. Soluble sulfatases such as arylsulfatase A (ARSA), ARSB, Iduronate 2 sulfat ase, sulfamidase, galactose 6 sulfatase, N acetyl galactosamine 4 sulfatase, and glucosamine sulfatase are targeted to lysosomes upon translation and act in acidic pH. Membrane bound members such as ARSC or steroid sulfatase (STS) ARSD, E, F, G, H, I ,J, K reside in endoplasmic reticulum and Golgi network and act in neutral pH (Parenti et al., 1997) The x ray crystal structures for three human sulfatases, ARSA, ARSB and STS have been resolved (Bond et al., 1997; Lukatela et al., 1998; Hernandez Guzman et al., 2003) They show a high degree of similarity with their N termini containing the acti ve sheet. The C terminal helix. The substrate binding pocket contains a divalent cation, usually calcium, and a highly co nserved cysteine that is indispensable for enzyme activity. These similarities suggest a common mechanism for catalysis. The tertiary structure of STS contains a transmembrane domain consisting of two helices about 40 in length containing hydrophobic residues. This domain is present in ARSA and ARSB also but is not hydrophobic as in STS, perhaps because they are soluble proteins. This transmembrane domain in STS has 12 cysteines forming ar the lipid protein interface presenting the hydrophobic residues to the membrane. In the lumen of the endoplasmic reticulum STS has the globular polar domain containing the catalytic site which is
24 identical with those in ARSA and ARSB. Crystal structure of STS with its two domains is shown in Figure 1 5. The conserved cysteine (Cys 75 in STS) residue in the active site is post translationally modified into a formylglycine (FG) which is further activated into hydroxyformylglycine (HFG) by a water molecule. HFG is crucial for the mechanism of hydrolysis by sulfatases and along with positively charged side chains of Lys 134, Lys 368 and Arg 79 that form the polar binding pocket. His 136 and His 290 also play important roles in the catalysis and charge neutra lization in the active site cavity. Steps in the catalytic mechanism are shown in Figure 1 6. Briefly, one of the hydroxy groups on HFG upon activation by Ca 2+ in the active site attacks the sulfur of the conjugated substrate. The covalent linkage of the s ulfur to the HFG causes the release of the unconjugated p roduct while the second hydroxy group of the HFG is involved in a nucleophilic attack on the ester bond releasing the HSO 4 moiety. The covalently linked sulfur has been observed in STS crystals conf irming the formation of sulfate ester of the HFG (Ghosh, 2007). Multiple sulfatase deficiency (MSD) is rare autosomal disease that results from decreased activity in all sulfatases due to mutations in the gene encoding the enzyme formyl glycine generating enzyme that activates sulfatases by converting the catalytic cysteine to formyl glycine (Cosma et al., 2003; Dierks et al., 2003; Dierks et al., 2005) Steroid sulfatase is the only sulfatase whose natural substrate and metabolic role has been completely understood (Ballabio and Shapiro, 2001). As the name indicates, it can hydrolyze alkyl and aryl steroid sulfates to generate their precursors. Important examples of such substrates are dehydroepiandrosterone sulfate (DHEAS) and estrone
25 sulfate (E1S), and the STS activity makes available their precursors which have been implicated in breast and endometrial cancer proliferation. Deletion of the STS gene or its flanking regions in the short arm of the X chromosome resul ts in X linked ichthyosis, one of the most common inborn errors of metabolism (Shapiro et al., 1989) STS is highly expressed in the placenta. Biochemica l and immunohistochemical studies have detected its presence in other reproductive tract tissues such as endometrium, prostrate, testis and ovaries along with breast, adrenal, brain, kidney and bone tissues. STS is also expressed in other parts of the body albeit in small quantities (Reed et al., 2005 ) Synergistic action of cytokines IL 6 and TNF activity of STS, possibly by posttranslational modification of the cysteine in the active site to formylglycine or by facilitating indirectly the uptake of the hydrophilic sub strate by changing transporter and membrane characteristics of the cell (Purohit et al., 1996; Purohit et al., 1997; Newman et al., 2000) Exposure to steroids can also activate STS as observed in testosterone treat ed male rats (Lam and Polani, 1985) and with increase in estrogen levels in pregnant guinea pigs (Moutaouakkil et al., 1984) Estrogens and Breast Cancer Estradiol is the most potent of the female sex hormones and acts by triggering the transcription of genes respons ible for its various cellular activities. Prominent among its numerous effects are its role in the female reproduction, sexual development and maintenance of bone integrity. About 95% of both pre and post menopausal breast cancers are hormone dependent i n the initial stages and estradiol has a crucial role in their progression and development (Lippman et al., 1986; Henderson et al., 1988) The relationship between higher levels of estrogens and higher incidence of breast cancer has been well established (James and Reed, 1980; Bernstein and Ross, 1993)
26 Estrogens can be carcinogenic in three main ways. Firstly, by the estrogen receptor mediated hormonal activity which results in activation of proto oncogenes and oncogenes such as c fos and c myc (Pasqualini and Chetrite, 2005) The second mechanism is by cytochrome P450 mediated metabolic activation of estradiol into nucleophilic substances capable of causing mutations in the cellular proteins. Thirdly, by the induction of ane uploidy (Page et al., 1985) Cholesterol is modified in the human body to generate various steroid hormones. Dehydroepiandrosterone (DHEA), a downstream product of cholesterol modific ation, in its sulfonated form is the second most abundant steroid (0.045 to 0.347 mg/100 m L ) after cholesterol (168 to 256 mg/100 m L ) in plasma and acts as a portable reservoir for the generation of sex steroids at different tissues of the body (K eys et al., 1950; Orentreich et al., 1984) hydroxysteroid dehydrogenase HSD) can form androstenedione which can in turn form estrone by the action of the enzyme aromatase. Estrone and estrad HSD enzyme catalysis in the tissues. Biotransformation of estrogens is depicted in (Figure 1 7). Estrone and estradiol can be sulfonated by SULTs and the sulfonated forms are not only amenable to excretion owing to their water solubility but also to transport to tissues via plasma. Reports indicate higher estradiol levels in malignant tissue compared to normal tissue around it (van Landeghem et al., 1985; Thijssen et al., 1987) Ti ssues like breast contain all the enzymes necessary to generate estradiol from circulating steroid sulfates. The two main ways of 17 E2 generation in the breast are named after the main enzymes involved as: aromatase pathway which involves the generation of
27 E2 from androgens and the sulfatase pathway in which the sulfatase enzyme converts the estrone sulfate, the major circulating estrogen, to estrone which is further converted to E2 This localized generation of E2 is critical in understanding the etiology of breast cancer in post menopausal women since ovaries are, in principle, incapable of producing estrogens in them (MacDonald et al., 1978; Reed et al., 1979) Treatment of hormone dependent breast cancer has been done for decades by managing synthesis of estrogens and their actions (Geisler and Lnning, 2005) Antiestrogenic drugs, such as tamoxifen, have been the first line drugs for the 80s and 90s. They exert their action by competing with estrogens for the estrogen rece ptor and thereby obviating the hormonal signaling. Recently the role of the first line drugs in metastatic and hormone dependent breast cancer has been taken by aromatase inhibitors that arrest the synthesis of estrogens from the androgens in the cancer ti ssue (Geisler and Lnning, 2006) The shift in the focus of treatment of cancer from inhibition of estrogen binding to estrogen synthesis suppression has led to investigati ons in the field of sulfatase inhibition. The reports of higher estrone sulfate concentration in tumoral tissue compared to normal tissue (Chetrite et al., 2000) and the intense estrone sulfatase activity in malignant brea st cancer compared to normal breast tissue validate such focus (Naitoh et al., 1989; Pasqualini et al., 1996; Pasqualini et al., 1997) It has also been reported that sulfatase activity is 40 500 times higher than t hat of aromatase activity in breast cancer tissue (Santner et al., 1984; Pasqualini et al., 1996) Sulfonation can cause a decrease in the cell proliferation effect of estradiol as its sulfate is incapable of bind ing to the estrogen receptor on the cell surface. Increase of sulfonation along with decrease in the activity of sulfatase enzyme therefore can be
28 helpful in breast cancer. It has been shown that patients with higher sulfatase mRNA expression have signific antly shorter disease free survival rates compared to ones with lower expression (Utsumi et al., 1999) P roduct Switching and Stimulation of Overall Catalysis by C elecoxib SULT2A1 is one of the three major SULTs that bring about 3 sulfonation of E2. It has been reported that in addition to E2 3S formation, it can also generate the 17 sulfate ( E2 17 S) of E2 as a minor product, which makes it unique among the SULTs active towards E2 (Wang and James, 2005) .The rate of E2 3S formation by SULT2A1 was found to be 8.8 fold higher compared to that of E2 17S. Interestingly, in the presence of a cyclooxygenase 2 (COX 2) selective drug celecoxib, the major product of 1 E2 sulfonation by recombinant SULT2A1 was modulated in a concentration dependent manner to increase E2 17S formation and decrease the E2 3S formation simultaneously such that the ratio of E2 17S/ E2 3S was 16 for the highest concentra Similar modulation of catalysis was seen with human liver cytosol as the source of the enzyme, bringing the E2 17/ E2 3S concentration to a maximum of 1. In a previous study by Cui et al. (Cui et al., 2004) celecoxib switched the major product of ethynyl estradiol (EE) sulfonation by recombinant SULT2A1 and liver cytosol from 3 sulfate to 17 sulfate. At a concentra overall catalysis by 3 to 4 fold compared to the controls. Also, the fact that different 50 value for celecoxib inhibition of its sulfonation coupled wi th the observation that the apparent kinetic constant for 17 sulfonation in the presence of celecoxib decreased by 2 fold at higher concentrations of EE made the authors speculate that the switching is due to
29 heterotropic modulation of SULT2A1 by celecoxib SULT2A1 with celecoxib bound at a heterotropic site assumes a conformation that is amenable to 17 sulfonation of EE and this heterotropic site is different from the substrate binding pocket. Sulfatase enzyme is capable of converting 17 E2 3S to E2 in the breast cancer tissue along with converting estrone sulfate to estrone, a precursor of E2 Interestingly it has been found to be incapable of converting estradiol 17 sulfate (E 17S) to estradiol (Pasqualini et al., 1989; Chetri te et al., 2000). Increasing the generation of E2 17S, a minor product of E2 sulfonation can be hypothesized to have a beneficial role in breast cancer treatment. Hypotheses and S pecific A ims The hypothesis and specific aims for the current researc h listed below follow from the observations mentioned above regarding the celecoxib modulation of estradiol sulfonation Hypothesis 1 Celecoxib interacts with h uman SULT2A1 to affect sulfonation of steroids. Specific aim 1 To determine the relationship bet ween structures of some steroids analogous to estradiol and the ability of celecoxib to modulate their sulfonation patterns catalyzed by recombinant SULT2A1. Specific aim 2 To investigate if the celecoxib effect is selective to SULT2A1 or if it could be r eplicated in mammalian model animals which contain enzymes analogous to SULT2A1, sheep and rat.
30 Specific aim 3 To understand the mechan ism of celecoxib modulation of SULT2A1 by molecular modeling studies by conducting protein ligand docking studies with av ailable crystal structures of the enzyme. Hypothesis 2 Small molecules encountered as constituents of food or cosmetic preparations can selectively inhibit SULTs that metabolize steroids. Specific aim here is t o test the result of specific inhibition of SULT1A1, SULT1E1 and SULT2A1 by triclosan in human liver cytosol upon estradiol sulfonation in the presence of celecoxib Hypothesis 3 Small molecules that inhibit SULTs can also inhibit steroid sulfatase enzyme. Specific aim here is t o study the effect of inhibitors tric lo san and celecoxib on the activity of sulfatase enzymes in mammalian placental microsomes.
31 Figure 1 1 Depiction of constituent p arts of the name of SULT enzyme a ccording to the nomenclature proposed in (Blanchard et al., 2004)
32 Figure 1 2 The expression values for each human sufotransferase enzymes in four different tissues: liver, small intestine, kidney, and lung. All numbers are percentages of total SULTs in respective tissues. Data from (Riches et al., 2009)
33 Figure 1 3 SULT2A1 structure shown with docked DHEA and PAP. Strands are shown in yellow, helices in red and loops in green. DHEA and PAP are shown as molecular surfaces in cyan a nd magenta respectively. Figure prepared using the PDB ID 1 J9 9 for SULT2A1 and DHEA co ordinates matched with those of PAP from PDB ID 1EFH.
34 Figure 1 4 A proposed (Negishi et al., 2001) sulfonation mechanism mediated by SULTs. Residue numbers correspond to hu man SULT1E 1 Substrate (ROH) is shown along with the co substrate PAPS and the three crucial amino acids His 107, Ser 137 and Lys 47.
35 Figure 1 5 Crystal structure of STS. PDB ID 1P49, reported in (Hernandez Guzman et al. 2003)
36 Figure 1 6 Catalytic mechanism for steroid sulfatase The modifications of the crucial amino acid residue, Cys 75 to give formylglycine (FG), hydroxylformylglycine (HFG) and formylglycine sulfate (FGS) in three stages of catalys is are also shown.
37 Figure 1 7 Estradiol generation in tissues from the transport forms of steroids. Aromatase and s ulfatase pathways are indicated in orange and white arrows, respectively
38 CHAPTER 2 CELECOXIB EFFECT ON ESTRADIOL ANALOGU E S ULFONATION Introduction Sulfonation apart from being a mechanism for elimination of lipophilic xenobiotic compounds serves as a process by which endocrine effects of steroid and thyroid hormones are facilitated. These endogenous compounds when sulfonated a re hydrophilic enough to be transported by plasma to their tissues of action (Loriaux et al., 1971; Robertson and King, 1979; Baulieu, 1996) In the case of steroid hormones, studies have shown that these sulfate co njugates are hydrolyzed by membrane bound enzyme steroid sulfatase to generate their precursors locally in the tissues (Martel et al., 1994) The importance of estrogens in the etiology of breast cancer has been well established (Ja mes and Reed, 1980; Page et al., 1985; Bernstein and Ross, 1993; Pasqualini and Chetrite, 2005) Findings that estradiol (17 E2) can be generated in breast cancer tissue locally via the sulfat a se enzyme hydrolysis of estrone sulfate (Vignon et al., 1980; Pasqualini et al., 1986; MacIndoe, 1988; Pasqualini et al., 1989a; Pasqualini and Chetrite, 2005) or aromatization of androgens (MacDonald et al., 1978; Abul Hajj et al., 197 9; Reed et al., 1979; Lipton et al., 1987) prompted research focus on the enzymatic systems involved in its generation and elimination. It is also known that the main estrogen sulfonating enzyme in humans, SULT1E1 is repressed in most breast cancer cell l ines compared to normal epithelial mammary cells (Falany and Falany, 1996) The impaired ability of the cancer cell to sulfonate 17 E2 when coupled with the fact that sulfatase enzyme activity is intense in malignant breast cancer tissue
39 compared t o normal breast tissue (Naitoh et al., 1989; Pasqualini et al., 1996; Pasqualini et al., 1997) place it at greater risk of estrogen mediated carcinoma. Celecoxib, a cox 2 inhibitor drug switched the concentration o f the major product of 17 E2 sulfonation from 3 sulfate to 17 sulfate in a concentration dependent manner by modulating the activity of the enzyme SULT2A1 (Wang and James, 2005) This switching, observed in human liver cytosol and also with the recombinant SULT2A1 enzyme, can be beneficial in the breast cancer as E2 17 sulfate is not a substrate for h ydrolysis by steroid sulfatase enzyme (Pasqualini et al., 1989b; Chetrite et al., 2000) Celecoxib was also shown to be capable of switching the product concentration of ethynyl estradiol previously and a heterotr o p ic modulation of SULT2A1 was proposed to be its mechanism of action as different substrate concentrations (0.39 to 2.5 M) had the same IC 50 value for inhibition of its 3 sulfate. The modulatory effect of celecoxib on E2 sulfonation prompted similar enzyme modulation studies on substrates analogous to estradiol with SULT2A1 Such a study was postulated to be helpful in investigating if the effect extended beyond E2 to substances that we re structurally similar to it. The substrates studied included ring B saturated estrogens, ring B unsaturated estrogens and a ring C unsaturated estrogen, 9 D ehydroestradiol (9 D E2). Structures of these analogues are shown in Figure 2 1. Among the ring B saturated estrogens tested estrone (E1 ) and 3 M ethoxyestradiol (3Me E2) contain just one hydroxy group (3 OH in estrone OH in 3 Me E2) E2 OH and 3 OH) and 2 H ydroxyestradiol (2OH E2) and 4 H ydroxyestradio (4OH E2) are catechol estrogens (CEs) with three hydroxy groups each (2 OH and 4 OH additional to those in E2 respectively) 1
40 dihydroequilenin ( Eqn) and 6 Dehydroestradiol (6D E2) were the ring B unsaturated estrogens. Except for E1 and 3Me E2 all other substrates contain both 3 OH (Figure 2 1). T hree different concentrations (50, 200 and 40 0 nM) of 17 E2 were studied to learn the effect of the substrate concentration on the modulation by celecoxib. T o add to the knowledge of the effect celecoxib has on overall sulfonation of E2 its inhibitory effect on the sulfatase enzyme was investi gated in the rat placental microsomes. Materials and M ethods Bacterial E xpression of SULT2A1 E. coli XL1 Blue cells transfected with the pKK233 2 vector containing the cDNA of the SULT2A1 enzyme were grown in an ampicillin (200 g/m L ) containing LB agar me dium and a single colony from this was introduced into 2L Luria broth. The cells were 600 value of 0.5 and then the expression is induced by adding 0.5 mM iso pr o pyl beta D thiogalactopyranoside (IPTG) to it. The medium was shaken overnight and then the cells were pelleted by centrifuging at 4200 rpm for 15 min. The pellet was resuspended in a lysis buffer containing 0.75 M Tris (Ultra pure, ICN), 0.25 M sucrose (Sigma 99+ %), 0.25 mM EDTA (Sigma grade) and 0.02 m g/m L lysozyme and cooled on ice for 20 min before centrifuging again. The resultant pellet was resuspended in 10 mM TEA buffer (Sigma 99.5%), pH 7.4 containing 10% glycerol (Sigma, min. 99%), 1.5 mM dithiothreitol (Bio Rad), 10 g/m L phenylmethylsulfonyl f luoride (Fluka) and sonicated 4 times in 10 sec bursts each time followed by 30 sec cooling to disrupt the cell membranes which allows the cytosolic protein to be separated upon subsequent centrifugation at 150,000 g for
41 1hr. The cytosol obtained was store d at 80 was purified by Milli Q TM water system to 18 Partial purification of the expressed enzyme was achieved in a process involving ion exchange chrom atography. The cytosol was applied to a 25 cm X 2 cm DEAE cellul ose (diethylaminoethyl cellulose) column which was equilibrated with the TEA buffer ( described above) at a rate of 25 m L /hr After a wash of 200 m L protein was eluted with 600 m L of 0 250 mM NaCl linear gradient and collected in 6 m L fractions. The protei n containing fractions from this experiment were identified by ultraviolet analysis at 280 nm and the presence of the desired enzyme among these was confirmed by enzyme assay using DHEA as the substrate. SULT2A1 A ssay For the experiments designed to study the effect of celecoxib, reaction mixtures estradiol) were incubated with 0.1 M Tris Cl (Ultra pure, ICN) pH 7.4, 5 mM MgCl 2 (99.3% pure, Fisher), partially pure SULT2A or catechol estrogens others) and 20 reaction was stopped by adding 0.3 m L of ice cold methanol to flocculate the protein and the supernatant was stored at 80 C until LC/MS/MS analysis. LC MS/MS A nalysis LC MS/MS analyses were performed using an Accela high speed LC system coupled with Finnigan TSQ Quantum Ultra triple quadrupole mass spectrometer equipped with heated electrospray ionization (H ESI) interface operate d in negative ion mode detection. The chromatographic separation of the 25 L reaction mixture injections was achieved with a BETASIL Phenyl (150 mm x 2.1 mm x 5 m) column
42 (Thermo Electron) with 50% acetic acid ammonium acetate buffer (0.1% CH 3 COOH, 5 mM CH 3 COONH 4 ) and 50% methanol at a constant flow rate of 0.2 m L /min. High pressure nitrogen (Airgas, Gainesville, FL, USA) was used as both sheath and auxiliary gas in the Finnigan TSQ Quantum Ultra instrument. The vaporizer and the ion transfer heated capil lary temperatures were mai respectively. The negative ion mode spray voltage was set at 2.5 kV. Ultra high purity argon (Airgas, Radnor, PA, USA) at a pressure of 1.5 mTorr was used as the collision gas in the second quadrupole of the mass spectrometer which was the collision cell. Steroid sulfates were measured in selected reaction monitoring (SRM) mode, monitoring the appropriate fragmentation transition of each target compound. The most sensitive of the isomer specific SRM transitions for steroid 3 s ulfates and steroid 17 sulfates were [M H ] [M SO 3 ] (m/z 271.17 for E2 ) and [M H] [HSO 4 ] (m/z 96.6) respectively. The optimal collision energy for each SRM transition was determined from collision induced dissociation and energy resolved mass spec trometry experiments. The H ESI interface operational parameters were optimized to achieve maximum sensitivity for [M H] of the compounds studied. The LC MS/MS system was controlled by Xcalibur (v.2.0) software, a flexible Windows NT PC based data acquisi tion system that allows complete instrument control. Effect of Celecoxib on Sulfatase Activity The sulfatase assay was done with 4 methylumbelliferysulfate as the substrate which yields 4 methylumbelliferone as the product of hydrolysis. The source of sulf atase enzyme was rat placental microsomes. The effect of celecoxib on the reaction was studied by adding two different concentrations of celecoxib to the reactions tubes with known concentration of 4 methylumbelliferylsulfate. For this a solution of 0.4 mM 4
43 methylumbelliferylsulfate was made in water with 0.05 M Tris H Cl and was added with of any celecoxib was also studied at the same concentration of 0.4 mM 4 methylumbelliferylsulfate with just the solvent DMSO and this served as t he control. Finally a tube identical to the control was maintained with no incubation to serve as a blank. The reaction was stopped by adding 2 m L methanol to the tubes. The protein in the tubes, at this point was allowed to flocculate for 10 min before th ey were centrifuged for 10 min at 2,000 rpm. The supernatants were analyzed for fluorescence immediately after adding 0.5 m L 1M Tris base to bring the solution to pH 10. A standard curve was obtained by using solutions of 4 methylumbelliferone with concent rations from 0.01 nM to 1 nM. Results Estradiol Analogue Sulfonation in the Presence of Celecoxib E2 concentrations ( 50, 200 and 400 nM ) showed a similar trend of generating more 17 sulfate with increasing celecoxib concentration with a concomitant decrease in amounts of 3 sulfate formed (Figure 2 2 ). One difference between 50 nM 17 E2 and higher concentrations of 17 E2 was that the point where the curves for 3 and 17 50 nM 17 E2 higher concentration of 17 E2 Another difference was that at 400 nM 17 E2 total sulfonation was inhibited to below the control tube levels at 80 M celecoxib but not at l ower 17 E2 concentrations. Among the 17 E2 analogues studied, E1 and 3 Me E2 have only one OH group (phenolic 3 OH in E1 and alcoholic 17 OH in 3 Me E2 ) and so yielded just one sulfate
44 each (Figure 2 3 ). Even though there is no significant difference (mea sured by test) among the values for the E1 sulfonation t he amount of E1 sulfate formed increased slightly with celecoxib concentration and reached a maximum at In the case of 3 Me E2 the sulfate formation was stimulated in the presence of celecoxib and there was at least 10 12 fold control. 17 E2 is unique among the analogues tested because it formed only 17 sulfate despite having two hydroxy groups ( Figure 2 3 ). The 17 hydroxy group of 17 E2 was sulfonated to a gr eater extent than the 3 hydroxy group of 17 E2 in the absence of celecoxib. Sharp Inhibition of this sulfonation was brought about by celecoxib concentrations beyond 50 M. All t he other a nalogues with two hydroxy groups, a phenolic one at 3 position and a 17 hydroxy generated both the sulfates in the presence of SULT2A1 (Figure 2 4). In the control experiments, the amounts of 3 and 17 sulfates formed were similar for all the substrates, but with increasing concentrations of celecoxib there we re some observable differences. 6D E2 and Eqn showed a pattern seen before with 17 E2 wherein, with increasing celecoxib concentration the amount of 3 sulfate generated decreased and that of 17 sulfa te increased. The formation of Eq 17 sulfate also is stimulated as in the case of 17 E2 albeit to a lesser extent There is no significant decrease in the amount of Eq 3 sulfate formed within the range of celecoxib concentration tested (0 80 M). For all three analogues there was stimulation of total sulfates generated in the presence of celecoxib, approximately 2.5 times for Eq and about 5 times 6D E2 and, Eqn compared to their controls.
45 9 D E2 sulfonation also generated both the possible sulfates at 3 a nd 17 poitions. The amounts of sulfates formed was switched in the presence of celecoxib, changing the major metabolite from 3 sulfate to 17 sulfate in concentration dependent manner (Figure 2 4 ). As in the case of Eq, the 9D E2 3 sulfate formation was not changed significantly by 0 80 M celecoxib Formation of 17 sulfate increased linearly with celecoxib concentration within the range tested. The stimulation of overall sulfonation was about 10 times compared to the control at 8 0 M celecoxib There were s ome significant differences in the sulfonation of the two CEs (Figure 2 5) The sulfates formed at the 17 hydroxy could be differentiated from those formed at the phenolic 2 OH, 3 OH and 3 OH, 4 OH for 2 OH E2 and 4 OH E2 estradiol, respectively by the LC r etention times, but identity of individual phenolic sulfates cannot be ascertained by the me thod of detection employed by here In the description below, it will be assumed that the major phenolic sulfate is the 3 sulfates for both the CEs and the minor ph enolic sulfate will be referred to as 2 sulfate for 2 OH E2 and 4 sulfate for 4 OH E2 In the con trol tubes with no modulator, 4 OH E2 formed more of the major phenolic metabolite, 3 sulfate whereas 2 OH E2 was not sulfonated to any great extent. Increasing c oncentration of celecoxib had an effect of increasing the 17 sulfates of both the CEs. Much of the increase in 17 sulfate in the case of 4 OH E2 came at the expense of decreasing 3 overa (Figure 2 5 ). With 2 OH E2 the overall sulfonation was considerably stimulate d by
46 celecoxib. The amount of 2 OH E2 17 is abou t 3.5 times the total sulfonation in the control. Effect of Celecoxib on Sulfatase Activity Celecoxib at the concentrations of 50 M and 100 M did not inhibit the sulfatase enzyme activity toward 0.4 mM 4 methylumbelliferysulfate. The activity of sulfatas e enzyme observed in the experiment, without celecoxib (6.2 nmol/min/mg), did not differ significantly from the activities obtained with 50 M (6.11 nmol/min/mg) or 100 M (5.3 nmol/min/mg) celecoxib. The values given are averages of duplicate measurements Conclusions and Discussion SULT2A1 is one of the major sulfotransferases in humans and is known to predominan tly sulfonate aliphatic hydroxy groups like the 3 OH in DHEA. Apart from hydroxysteroids, SULT2A1 is also known to sulfonate endogenous phenoli c hydroxy groups on estradiol and estrone and the alcoholic groups on benzylic and allylic alcohols (Miller, 1994; Glatt et al., 1995; Wang and James, 2005) It is expressed mainly in adrenal glands, liver and jejun um (Comer and Falany, 1992; Her et al., 1996) DHEA not only plays a role in brain development and function (Baulieu and Robel, 1998; Compagnone and Mellon, 1998; Frye and La cey, 1999; Markowski et al., 2001) but is also the circulating steroid reservoir in humans as it can be hydrolyzed back to DHEA which can further be converted to testosterone and estrone by the actions of enzymes 17 HSD and aromatase respectively in ti ssues. Major amount of DHEA generated by the adrenal glands is secreted into the plasma (10 M in young adults) in the sulfonated form and a comparatively very small amount (10 nM) in its unconjugated form (Baulieu, 1996)
47 Two crystal structures of SULT2A1 have been reported in the literature, one co crystallized with DHEA (Rehse et al., 2002) and the other with inactive co substrate, PAP (Pedersen et al., 2000) which are helpful in understanding the molecular basis of its substrate specificity. The substrate binding pocket of SULT2A1 is reported to be large enough to allow two molecules of DHEA to ass ume alternate binding positions, one catalytic and the other non catalytic (Rehse et al., 2002) T he hydrogen bonding of the hydroxy group of the substrate whose proton is to be substituted with a sulfonate group from PAPS, with the His 99 of the enzyme during catalysis, seems to be critical as is the case with other SULTs such as SULT1E1 (Kakuta et al., 1998b) Among substr ates studied here with j ust one hydroxy group, E1 does not have a hydroxy but the observation that the 3 sulfonation was not inhibited until the 3 sulfonation of 17 E2 was inhibited by more than 80% by the same concentration of celecoxib (Wang and James, 2005) A possible reason for this could be that the absence of the formation of the hydrogen bond between the 17 keto group and His 99 mandates that the E1 molecule assume a conformation amenable for 3 sulfonation. 17 sulfonation of 3 Me E2, on the other hand started to saturate on ly with the highest concentration of celecoxib tested, possibl y due to the fact that there was no 3 OH group competing for the His 99 It was previously found that the capacity ( k cat /K m ) of SULT2A1 for Z enantiomers of Hydroxytamoxifen was higher than th at for E enantiomers suggesting isomer preference (Apak and Duffel, 2004) The formation of 17 sulfate of 17 E2 by SULT2A1 at a rate higher than that of 3 sulfate of 17 E2 supports this observation.
48 The estradiol analogues, on the whole, showed behavior similar to 17 E2 upon celecoxib modulation. Although the patterns of switching, evidenced by the cross over points of the individual sulfate curves differed between the substrates, the fact that switching occurred was unmistakable for all the substrates with 3 OH groups. Two of the substrates with two hydroxy groups tested, Eq and Eqn are found as sulfates in the pregnant mare urine and are used in estrogen replacement therapy in menopausal women as conjugated equine estrogens (Bhavnani, 2003) Another ring B unsaturated analogue 6D E2 and the only ring C unsaturated estrogen tested here 9D E2 also produced more 1 7S, showing the celecoxib effect. The role of estrogens in carcinogenesis is chiefly by receptor mediated activation of cell proliferation (Nandi et al., 1995; Castles and Fuqua, 1996) Another route by which estrog ens can cause tumor progression is by forming catechol estrogens (CEs) that are in turn metabolized to quinones and semi quinones that are capable of forming carcinogenic DNA adducts (Bolton and Shen, 1996; Yager and Liehr, 1996; Cavalieri et al., 1997) O methylation of the CEs by catechol O methyltransferase (COMT) is one major pathway for detoxification apart from sulfonation, which has an import ant role in steroid metabolism (Weinshilboum and Raymond, 1977; Wilson et al., 1984; Boudkov et al., 1990; Weinshilboum et al., 1999) Conflicting reports have suggested that an inherited defect of this polymorphic enzyme (Lavigne et al., 19 97; Thompson et al., 1998; Mitrunen et al., 2001) may put the subject under increased risk for the occurrence of breast cancer. Since the sulfate conjugation of these CEs is the second metabolic defense against their mutagenic potential after COMT, it is of vital importance in breast cancer prevention. The increased overall sulfonation of 2 OH E2 and the increase in the
49 share of 17S for 4 OH E2 sulfonation should help in urinary excretion of these compounds. The overall effect of celecoxib seems to be to mod ify the substrate binding pocket in such a way that the sulfonation of 17 hydroxy of the estradiol analogues is favored over the 3 hydroxy and the overall sulfonation is stimulated. The structure of the analogue seems to determine the extent of this switc hing and stimulation. 6D E2, Eqn and 9D E2, molecules with double bonds in conjugation with their aromatic A rings that makes them flatter compared to Eq, showed highest amounts of stimulation apart from 3Me E2. The limited stimulation in the case of CEs suggests a role for the aromatic ring and its substituents in the alternate binding that brings about the switching and stimulation of sulfonation. The di fference between the 17 hydroxy sulfonation of 17 E2 and 17 E2 is a case in point for the isomer specificity/selectivity of SULT2A1.
50 Figure 2 1 Structures of estradiol analogues tested for the effect of celecoxib modulation. Shown in red are the regions in each molecule that are differe nt from the corresponding ones in 17 Estradiol.
51 Figure 2 2 estradiol by SULT2A1 enzyme Two individual experiments were conducted for every celecoxib concentration and each of these reactions was analyzed by LC MS/MS in triplicate. Data points in the plots are Mean SD for all six measurements. Red circles represent 17 E2 3S and the green ones represent 17 E2 17S.
52 Figure 2 3 Effect of celecoxib estradiol and 3 Methoxyestradiol sulfonation by SULT2A1 enzyme (all substrates were 400 nM in concentration) .Two individual experiments were conducted for every celecoxib concentration and each of these reactions was analyzed by LC MS/MS in triplicate. Data points in the plots are Mean SD for all six measurements. Red circles represent 3 Sulfate of E1 and the green ones represent 17 Sulfates of 17 E2 and 3Me E2.
53 Figure 2 4 Effect of celecox sulfonation of 400 nM estradiol analogues by SULT2A1 enzyme Two individual experiments were conducted for every celecoxib concentration and each of these reactions was analyzed by LC MS/MS in triplicate. Data points in the plots are Mean SD for all six measurements. Red circles represent 3 S ulfates, the green ones represent 17 Sulfates and total sulfonation is shown as blue triangles
54 Figure 2 5 Effect of celecoxib on sulfonation of 400 nM catecho l estrogen sulfonation by SULT2A1 enzyme Two individual experiments were conducted for every celecoxib concentration and each of these reactions was analyzed by LC MS/MS in triplicate. Data points in the plots are Mean SD for all six measurements. Red c ircles represent 3 Sulfates, the green ones represent 17 Sulfates and the 2 and 4 Sulfates are shown as purple and orange diamonds re s pectively.
55 CHAPTER 3 SPECIES DIFFERENCES IN THE CELECOXIB EFF ECT Introduction The study that showed the swit ching of the major product of E2 sulfonation from E2 3 sulfate to E2 17 sulfate in the presence of celecoxib was conducted with human liver cytosol and recombinant human SULT2A1. No switching was observed in studies conducted previously with channel cat fish liver cytosol as the enzyme source (Wang and James, 2007) A study similar to these was conducted on two mammalian model animals, sheep and rat to observe the effect of celecoxib. Rat liver has been known to be sexually dimorphic for metabolic enzymes in general and it has been reported that phenol sulfotransferases are male specific (Liu and Klaassen, 1996b) whereas hydroxysteroid sulfotr ansferases (rat analogs of SULT2A1) are female specific (Liu and Klaassen, 1996a) So, three each of male and female rat liver preparation s were used for the study along with three female sheep liver cytosol. Materials and Methods Assay of R at and S heep Liver C ytosol The individual sheep and rat tissues were tested for the effect of celecoxib on estradiol sulfonation in reaction mixtures co mposed of 50 nM [3H] E2 50 mM Tris Cl (pH 7.4), 5 mM MgCl 2 20 M PAPS and either 140 g of sheep liver cytosolic protein or 75 g of male rat or 20 g of female rat liver cytosolic protein. A s olution of ce lecoxib in DMSO was added to give a final c oncentration of 100 M concentrations in the tests and the controls F inal concentration of DMSO was 1% v/v in both tests and controls Test and control reactions that were not incuba ted were designated as blanks. After the
56 e reactions were stopped with 0.3 m L methanol and kept on ice for 20 min to flocculate the protein. Centrifugally separated supernatants were then filtered and analyzed by HPLC. HPLC A nalysis A Beckman Gold Nouveau HPLC system equipped with UV and fluores cence ram IN/US Systems Inc., Tampa, Florida) was used for the analyses. A C 18 reverse phase column (4.6mm x 25cm) with a C 18 pre column (Discovery system, Supelco, Bellefonte, PA) at a constant flow rate of 1m L /min with 0.005M Pic A (tetrabutylammonium sulfate) in 55% methanol was used. The scintillation cocktail flow for the radiochemical detector (In flow 2, IN/US Systems, Supelco, Bellefonte, PA) was maintained at 3 m L /min. E2 E2 3S and E2 17 S were identified by the retention times of 26 min, 1 4 min and 1 2 min respectively. Results Sheep and M ale R at The amount s of protein for sheep and rat were chosen so as to have not more than 50% estradiol conversion to sulfates. The celecoxib concentrat ion tested, 100 M, has been shown to cause maximum product switching from E2 3S to E2 17S in human liver cytosol previously. In the sheep and male rat cytosolic preparations however, celecoxib inhibited the amount of sulfates formed, regardless the position of sulfonation. Figure 3 1 shows representative HPLC chromatogra ms of both sheep and male rat In control reactions in both the test animals E2 3S is the major product but a trace amount of E2 17S is also generated and the amounts of both the sulfates were reduced in the test reactions with 100 M celecoxib. Th e blanks with an d without celecoxib did not show any sulfonation. The activities reported in Table 3 1 and Table 3
57 2 (averages of two duplicate samples) show that there has been a marked reduction in the sulfotransferase activity upon celecoxib addition wi th respect to 17 E2 3S and E2 17S formation but do not show any significant switching of the concentration of the products formed E2 3S was the major product formed in the cases of both sheep and male rat with only a trace amount of E2 17S being produced. Th ough the activities with respect to 3 sulfonation and 17 sulfonation of E2 differ among individual samples studied, the inhibition upon celecoxib treatment is universal and no enhancement in the amount of E2 17S was observed either. No detectable a mount of E2 disulfate, a negligibly small product of E2 sulfonation in human liver cytosol, was found. Female R ats In the case of the control female rats E2 17S was the major product of sulfonation of E2 (Figure 3 2) This is in contrast to the male liver sulfonation where E2 3S is the major form. There is some inhibition (especially in rats 1 and 3, Table 3 3 position of sulfonation. Discussion The sele ctive COX 2 inhibitor celecoxib did not show the effect of switching relative concentrations of the products of E2 in sheep and male rat liver cytosolic preparations as it does in the case of humans. It was reported that out of 5 major human SULT isofo rms studied only SULT2A1 forms E2 17S as a product of E2 sulfonation and it was inferred that the action of celecoxib in increasing the concentration of E2 17S was by acting on the enzyme in a way to change its conformation so as to facilitate the production of 17S and not 3S product (Wang and
58 James, 2005) This alloster ic inhibition of 3S production is clearly not found in the sheep and male rat samples. The m ost interesting finding of this study is that the major product of 17 E2 sulfonation with female rat liver cytosol was E2 17S however celecoxib did not increase the rate of 17 Sulfonation. Sexual dimorphism of the rat liver and the presence of low amounts of hydroxysteroid sulfotransferase in male rat livers could e xplain the inhibitory effect of celecoxib in the male rat experiments. Celecoxib was found to be inhibitory to SULT1A1 in humans (Wang and James, 2005) analogues of which are the predominant forms of SULTs in male rat liver (Liu and Klaassen, 1996b) These species differences mean that the celecoxib effect on estrogen sulfonation cannot be studied in these animals. Even though the human and rat hydroxysteroid SULTs have DHEA as their principal endogenous substrate, they have been known to show differences in their activities t owards various other substrates. In a study comparing the activation of benzylic alcohols into mutagens it was found that human enzyme was 67 fold more active than the murine one towards 1 (1 pyrenyl) ethanol (Glatt et al., 1995) On the other hand, for 7 hydroxymethyl 12 methylbenz[a]anthracene, rat enzyme was 27 fold more active than the human (Glatt et al., 1995) hydroxytamoxifen, a metabolite of antiestrogenic drug tamoxifen, only one was a substrate for rat hyd roxysteroid SULT whereas the other three were inhibitor s (Apak and Duffel, 2004) H uman SULT2A1 on the other hand was found to sulfonate all four stereoisomers in the same study
59 Structural differen ces between the substrate binding sites of these analogous enzymes reflective of differences in amino acid sequences most probably explain the differences in selectivity of substrates as discussed in Chapter 4
60 (A) (B) (C) (D) Figure 3 1 Representative chromatograms of sulfonation of 50 nM 17 E2 in the presence and absence of ce l e coxib. ( A) and (B) are sheep liver cytos ol without and with 100 M cel e coxib ( C ) and (D) are male rat liver cytosol without and with 100 M cel e coxib (n=2)
61 (A) ( B ) Figure 3 2 Representative chromatograms o f sulfonation of 50 nM estradiol by female rat liver cytosol with out (A ) and wit h (B (n=2)
62 Table 3 1. are averages of duplicate measurements. Female sheep sampl e 1 2 3 0 100 0 100 0 100 E2 3S activity (pmole/mg/min) 33.49 15.55 22.62 10.94 11.25 5.99 E2 17S activity (pmole/mg/min) 2.47 1.18 0.79 0.46 0.36 0.00 E2 17S/E2 3S* 0.07 0.08 0.04 0.04 0.04 -* E2 17S/E2 3S is the rati o of E2 3S activity and E2 17S activity shown in each column.
63 Table 3 2 averages of duplicate measurements. Male rat sample 1 2 3 0 100 0 100 0 100 E2 3S activity (pmole/mg/min) 17.56 4.86 17.42 5.28 26.18 8.18 E2 17S activity (pmole/mg/min) 0.99 0.00 1.32 0.43 2.03 0.80 E2 17S/E2 3S* 0.06 -0.08 0.08 0.08 0.09 E2 17S/E2 3S is the ratio of E2 3S activity and E2 17S activity show n in each column.
64 Table 3 3 averages of duplicate measurements. Fem ale rat sample 1 2 3 0 100 0 100 0 100 E2 3S activity (pmole/mg/min) 6.6 5.4 5.3 5.1 6.6 2.9 E2 17S activity (pmole/ mg/min) 31.7 24.7 22.4 22.0 31.4 25.6 E2 17S/E2 3S* 4.8 4.6 4.2 4.3 4.8 8.8 E2 17S/E2 3S is the ratio of E2 3S activity and E2 17S activity shown in each column.
65 CHAPTER 4 LIGAND DOCKING STUDI ES ON THE CELECOXIB MODULATION OF ESTRAD IO L ANALOGUE SULFONATION Introduction It has been shown previously that celecoxib switching of the concentration of the sulfonation products of 17 E2 is due to its modulation of SULT2A1 activity (Wang and James, 2005) The effect has been hypothesized to be due to allosteric modulation of SULT2A1 by celecoxib (Cui et al., 2004; Wang and James, 2005) and a molecular docking study (Yalcin et al., 2008) showed that it could indeed be so. This celecoxib effect has also been observed in various 17 E2 analogues as discussed in Chapter 2. A model for the effect of celecoxib on SULT2A1 activity that could help rationalize its effects on the sulfonation of these structurally diverse compounds can shed some light on the overall nature of the enzyme at the molecular level. Adding to the knowledge about this effect is the observation made in Chapter 3 that female rat liver cytosol catalyzes 17 s u l fonation of 17 E2, a minor product in male rat, sheep and human liver cytosolic experiments. The pursuit of the mechanism of the celecoxib effect, on 17 E2 in particular, is relevant give n that celecoxib has been found to effective in preventing mouse model breast cancer (Lanza Jacoby et al., 2003; Basu et al., 2004; Levi tt et al., 2004; Zhang et al., 2004) In light of the facts that celecoxib has been shown to be effective in preventing sporadic colorectal adenomas in people (Bertagnolli et al., 2006) and studies have reported in cre ased total estrogen and estrone (2 and 2.4 fold respectively) in colon carcinoma tissues than in normal colonic mucosa (Sato et al., 2009) it is imperative to parse the mechanism of celecoxib action. The sulfonat ion behavior of 17 E2 analogues observed in the presence and absence of celecoxib was studied further with the help of molecular modeling and ligand
66 docking studies. Compounds previously studied (unpublished) in our lab viz. dehydroepiandrosterone (DHEA), androstenediol (AD), epitestosterone (Epi T), testosterone (T) were added to the set of 9 compounds discussed in Chapter 2 in this study. The activities of SULT2A1 sulfonation for these compounds are given in Table 4 1. These non aromatic steroids were th e most active substrates for SULT2A1 with AD and Epi T being the best substrates tested by far, followed by DHEA and T in that order. The concentration range of celecoxib (0 80 M) tested with these compounds inhibited them slightly, but did not reach IC 50 values for any of them. In the case of AD, which has both 3 and 17 OH groups, only 3 Sulfate was generated with SULT2A1 indicating that an aromatic A ring is a necessity for product switching. Also the molecular basis for the generation of 17 E2 17 s ulfate by female rat liver cytosol in the absence of celecoxib was investigated with the help of a homology model. Method The crystal structure of SULT2A1 resolved in the presence of DHEA with the PDB ID 1J99 (resolution of 1.99 ) was used to generate a h omology model with the help of SWISS MODEL (Arnold et al., 2006; Kiefer et al., 2009) server to construct missing residues from the protein sequence under the ID NP_003158.2 in the NCBI database. Ligand docking stud ies along with receptor and ligand preparation and energy cal c u lations were conducted on Sybyl x 2.0 program running on a Linux operating system. Flexidock utility in Sybyl was used for the ligand docking. The SULT2A1 structure file was prepared for docking using the Biopolymer utility in Sybyl. The various steps carried out in the preparation of the protein included termini treatment to charge the terminal amino acid residues, adding hydrogen atoms adding Amber7 FF99 charges, fixing side chain amides and p erforming staged minimization in Amber7 FF99
67 force field (Case et al., 2005) in 100 steps. The non aromatic steroids were sketched in E2 from PDB ID 1AQY They were prepared by checking the Amber atom types on them, adding Gateiger Huckel charges and minimization steps. The crystal structure 1J99 ha s th e ligand, dehydroepiandrosterone (DHEA) with its 3 OH group in catalytic position (Rehse et al., 2002) For a ligand to be in catalytic position for SULT 2A1, its sulfonate acceptor has to be in hydrogen bonding distance from the conserved catalytic residue His (His 99 in human SULT 2A1). The liga nd molecules were placed in the binding pocket before the docking as Flexidock needs the ligand to be roughly in position in the active site to be able to generate the results. For docking a 10 area around the DHEA molecule in the crystal structure was s elected as the active site. The various steps involved in preparing to run Flexidock include removing the water in the active site, checking the atom types on the ligands and receptor, adding hydrogen atoms and charges to both the molecules. Only the bonds in the ligands were allowed to be rotate d The binding site was left rigid in both its backbone and side chains A total of 5000 generations were chosen as the limit for the docking experiment. Flexidock utilized a genetic algorithm in which number of generations specified for each run depends on the number of rotatable bonds in the experiment. The number of rotatable bonds plus six is called a gene and this number is multiplied by 500 1000 to get the number of required generations The algorithm s earches for favorable conformations starting with a random one and the desired characteristics (genes, in Darwinian sense) are passed on to the successive generation until a minimum is reached. Increasing the generations well beyond the specified minimum f avors a reasonable solution. But this
68 solution may not always be the catalytically active conformation required. So, different number of generations, starting with different random seed numbers are utilized in the preliminary experiments using all the liga nds until a suitable set of generations was identified for the given receptor. Flexidock gives up to 20 solutions (or conformations) along with their energies for each run. For results shown below, Flexidock run was performed for each ligand with 5000 gene rations and the conformation with catalytically viable conformation with highest free energy gain was taken as the solution. T he experiments were repeated six times for each ligand and the energies reported are the averages of all the experiments. The fi gures along with the solvent excluded molecular surfaces shown here were generated in Chimera (Sanner et al., 1996; Pettersen et al., 2004) Results It was observed before (Rehse et al., 2002) that DHEA can bind to SULT2A1 in two alternate binding modes, of which one is catalytic and the other non catalytic. The 3 hydroxy group of the catalytic mode is in hydrogen bonding distance to the conserved His 99 residue, a feature necessary for catalysis. This mode is oriented in such a way that the plane containing the two oxygen atoms on DHEA which are the farthest heavy atoms from each other in the molecule are oriented roughly in parallel to the largest opening of the binding site flanked by Tyr 238 and Trp 77 and the molecule is embedded deep into the binding si te. The catalytic mode is also stabilized by its van der Waals interactions with the side chains of Leu 234 and Met 137. The non catalytic mode on the other hand was reported to have hydrophobic interactions with many more residues viz. Phe 139, His 99, Tr p 77, Tyr 231 and Trp 72. The latter m ode is non catalytic despite it being more probable energetically as its 3 hydroxy group goes
69 deeper into the binding pocket making the hydrogen bond with His 99 unfavorable. The non catalytic mode is closer to one sid e of the pocket rather than in the middle of it and largest opening of the binding site. Dockings in the A bsence of C elecoxib Each one of the substrates has more th an one favorable binding and more often than not the conformation achieved by employing a large number of generations for docking (in the range of >10000 generations) resulted in a non catalytic binding with respect to the distance between the sulfon ate ac cepting group and the His 99 of the receptor. As likely as this conformation is in vivo and as useful as it may be to explain the comparatively lesser affinity and broad substrate specificity of SULT2A1 towards its ligands, it does not always explain the s ulfonation behavior of the individual substrates studied here. Moreover, Flexidock needs the ligand to be placed in the receptor prior to the docking run and the solutions it generates depend on the orientation of the ligand and the number of generations t he genetic algorithm is allowed. Hence the criteria for choosing the binding conformations of the substrates needed to be defined prior to the execution of the final docking procedure. The 3 hy d roxy groups of the ligands (3 keto group of T and Epi T and 3 Methoxy group for 3Me OH of DHEA co crystallized with SULT2A1 reported in the crystal (PDB ID 1J99) and the docking was conducted with a generation limit of 5000. The results generated (about 20 for each ligand) by Flexidock we re examined visually and the catalytic conformation with least energy was chosen as the solution. These steps were repeated several times to ensure the reproducibility of the results.
70 In the absence of celecoxib, the docked conformation of DHEA approximat ed closely that of the crystallized DHEA (Figure 4 1). The various ligands tested assumed conformations roughly similar to either the catalytic or non catalytic mode of DHEA bindings described above. DHEA and AD docked very s OH group i n catalytic position and their C 18 and C 19 groups facing the opening of the binding si te so as to avoid the clash of C 19 with Trp 134. The 17 hydroxy group of AD was not in a position to be sulfonated. Epi T and T docked in conformations very dissimilar to each other, reflecting their isomeric difference with respect to the 17 h ydroxy group (Figure 4 2). Epi OH assumed a more vertical orientation whereas T with OH was more horizontal. These two orie ntations were more similar to the non catalytic and catalytic bindings of DHEA respectively than to each other. E2, 9D E2 6D E2, Eqn, and Eq docked in conformations that favored 3 sulfonation. Except for the fact that the molecule itself is f lipped by E2 approximates the catalytic binding mode of DHEA the best among the compounds with two hydroxy groups (Figure 4 1). This match is reflected in it being the most sulfonated among t he estrogen analogues in the absence of celecoxib (Table 4 1) 9D E2 is not a very good substrate, even though it has a conformation similar to that of the catalytic DHEA as it goes too E2 and catalytic DHEA (Figu re 4 3). 6D E2, Eqn, and Eq were also poor substrates in the absence of celecoxib as the dockings show that their docked conformations do not lie in the zone of the substrate binding pocket occupied by the catalytic orientation of DHEA (Figure 4 3).
71 The c atechol estrogens were not good substrates of SULT2A1, and this is reflected by their control activities given in Table 4 1. The presence of the additional phenolic group in these molecules makes it difficult for them to assume the binding configuration of E2 His 99 in the binding pocket is flanked by two tyrosine residues, Tyr 160 and Tyr 231 whose side chain phenolic oxygen atoms are at distances of 5.4 and 6.0 respectively 99 respectively. The dockings of 4OH E2 and 2OH E2 in the absence of celecoxib (Figure 4 4 ) had to avoid the clash of the additional phenolic groups with these tyrosines and hence are different from that of E2 (Figure 4 1). The dockings support the hypothesis made before that the major metabolite in case of both the compounds was 3 Sulfate. To avoid the clash with Tyr 231 of its 4 OH group, 4OH E2 assumes a position that is similar to the non catalytic position of DHEA. To avoid a clash with Tyr 160 and to have its 2 OH group in a position similar to that of the 4 OH of 4OH E2, 2OH E2 has to assume the position shown in Figure 4 4 Two compounds that are exceptions to the rule that the docked conformation needs to be similar to that of the catalytic conformation of DHEA in order to be a good substrate E2 and E1 (Figure 4 5 ). Both these molecules seem to be assuming conformations similar to the non hydroxy group of the non catalytic conformation goes too deep into the pocket and is p hosphate group of the inactive co factor PAP than to His hydroxy E2 is perfectly positioned to be sulfonated as its axial oxygen atom is in hydrogen bonding distance to His 99. In the case of E1, an additional hydrogen bond between the side chain of Ser 80 and the acceptor 17 keto group keeps
72 E2 could explain why it assumes a totally different conformation despite of its obvious structural sim hydroxy group and the additional hydrogen bond can be demonstrated by the fact that 3Me E2 with a docked orientation E2 and E1 is a very poor substrate of SULT2A1 (Figure 4 5 ). Dockings in the P resen ce of C elecoxib The docked conformation of celecoxib in the binding site occupied the same area as the catalytic mode of DHEA in the crystal structure, as predicted by Yalcin et al previously. The trifluoromethyl group on the pyrazole ring goes farthest in to the pocket and leaving the sulfonamide on the outer side One of the fluorines of the trifl u o romethyl group and the nitrogen of the sulfonamide group are in hydrogen bonding distance to the side chain amine of Lys 44 and the backbone carbonyl group of M et 16 respectively. These interactions could anchor the celecoxib molecule to the side of the pocket leaving the phenylmethyl group floating in the middle, a conformation that can totally obstruct the binding of DHEA in the catalytic mode. Celecoxib bindi ng mode is shown in Figure 4 6 E2. In the presence of celecoxib the substrate binding pocket is truncated and the substrates can only assume a vertical orientation akin to the non E2, as shown in Figure 4 6 docks in the ve rtical position leaving its 17 hydroxy group in the catalytic position. The aromatic interaction between the A E2 and the phenylmethyl group of celecoxib along with the interactions with Pro 14, Phe 18, Trp 77, and Tyr 231 favor this binding. Figure 4 7 shows the overlap of the docked conformations of AD and DHEA in the E2 docking shown in Figure 4 6 The lack of the
73 aromatic interaction with the celecoxib and their molecular shapes that are far from being fla E2 render them incapable of forming the essential hydrogen bond with the His 99. T and Epi T both hav ing a non aromatic A ring and shapes more similar to E2 seem to assume conformations not ideal for hydrogen bonding with His 99 (Figure 4 8 ). The compounds 9D E2, 6D E2, Eqn and Eq have an aromatic A ring and all of them showed switching of the major product of sulfonation by SULT2A1. The docked conformations of these molecules, shown in Figure 4 9 E2 i n Figure 4 6 6D E2 and Eqn, the flattest of the set and those E2 in E2 in thei r conformations. 9D E2, with unsaturation in ring C conjugated with aromatic ring A is flat enough to assume a conformatio n that allows 17 hydroxy sulfonation even though its A ring is tilted a way from the phenylmethyl group of celecoxib. The compound that showed smallest amount of product switching and stimulation of overall sulfonation among the analogue E2 was Eq and its docked conformation could help rationalize these results. The unsaturation at the 7 position in ring B twists this molecule with respect to the other three molecules in such a way that it can have a better interaction with the ph enylmethy l group of celecoxib, perhaps better than the ot hers, but leaves its 17 hydroxy group farther away from His E2 that showed a docking conformation in the presence of celecoxib that allowed 3 sulfonation (Figure not shown). This interaction could explain the stimulation of Eq 3 sulfonation in the presence of lower concentrati o ns o f celecoxib.
74 The catechol estrogens also show the switching of the major product from 3 Sulfate to 17 Sulfate in the presence of celecoxib and they dock in configurations (Figure 4 10) very similar to E2 The stimulation of 17 Sulfate formation was also almost identical for these two compounds (as seen in Chapter 2, Figure 2 5). E1 and 3Me E2 have similar docked conformations in the presence of celecoxib. Both molecules have their aromatic rings in close proximity to celecoxib for the stacking interaction (Figure 4 11 ). This leaves the 17 keto group of E1 and the 17 hydroxy group of 3Me E2 in hydrogen binding distance to His 99, a scenario that can help rationalize the inhibit i on of E1 3 sulfonation and the stim ulation of 3Me E2 17 sulfonation observed E2, E1 and 3Me E2 but its ax ial hydroxy group at the 17 po s i tion is turned away from the His 99 making the hydrogen bonding interaction difficu lt to achieve (Figure 4 11 ). Rat vs H uman SULT The homology model of ST 60, which is the most abundant form of hydroxysteroid SULT in the female rat (Liu and Klaassen, 1996a) is similar in amino acid composition and the overall structure of the binding site when it is overlapped with the human SULT2A1 (Figure 4 1 2 ). The amino acids His 99, Trp 77, Tyr 160, Trp 72, Pro 43, Phe 18, Pro 14 in SULT2A1 have exact matches with the amino acids in ST 60. The differences in the crucial amino acids are: Gly 17 of SULT2A1 replaced by Trp 16, Met 16 by Phe 15 and the Tyr 238 by Leu 23 5 The difference of consequence seems to be the replacement of Gly 1 7 in SULT2A1 by Trp 16 in ST 60 as the hydrophobic side chain of this tryptophan is inverted into the pocket, thereby reducing its volume. This E2 found in SULT2A1 and allows only
75 the vertical orie ntation that has the 17 hydroxy group in the catalytic position. Trp 16 in ST 60 seems to be doing what celecoxib docking does to the binding site o f SULT2A1. Discussion Two crystal structures of human hydroxysteroid sulfotransferase have been reported, one co crystallized with the inactive co factor PAP (Pedersen et al., 2000) and the other with the endogenous substrate DHEA (Rehse et al., 2002) There is a crucial difference between these two structures pertaining to the access to the substrate binding site. The loop of amino acids from Try 231 to Glu 244 in the former truncates the substrate binding site whereas in the latter this loop is moved away from the opening of the binding site. I t has been observed that the order of binding of the substrate and co fa ctor will have an effect on the s ulfonation behavior of the enzyme. It has been shown that DHEA, the principal endogenous substrate of SULT2A1 can bind to it productively independent o f the PAPS binding but a larger molecule like raloxifene needs to bind prior to the PAPS binding for the full binding pocket to be available and for the sulfonation to proceed (Cook et al., 2010). SiteID analyses of both the available SULT2A1 crystal struc tures using Sybyl2.0 showed that there is no other site available except for the co factor and substrate binding sites for the docking of celecoxib. Only the crystal structure with DHEA has a binding pocket that can accommodate both celecoxib and a sulfona E2 and the truncated binding site in the other structure would not allow modeling of celecoxib in it as reported earlier (Yalcin et al., 2008). All the available crystal structure s of sulfotransferases co crystallized wit h PAPS or PAP in the Protein Data Bank (with PDB IDs 3U3J, 3U3K, 3U3M, 3U3O, 3F3Y, 3CKL, 3BFX, 2REO, 2Z5F, 2H8K, 2GWH, 2DO6, 2A3R, 1Q1Q, 1Q1Z, 1Q20, 1Q22,
76 1LS6, 1G3M and 1EFH) have near identical conformation of the PAPS (Figure 4 1 3 ) This is true despite the fact that the data set compared contains enzyme models that are less than 40% identical like SULT1A1 and SULT2A1. An attempt at modeling the PAPS molecule into the co factor binding site of the SULT2A1 model to mimic the conformation identical to the one in the crystal structure with PAP showed that it would clash with more than one residue in the pocket. A change in the conformation of the enzyme is indispensable for the sulfonation to proceed and for the PAPS to bind in catalytic mode as seen in Figu re 4 1 3 The crystal structure with bound DHEA when used for the docking experiments gave satisfactory explanations for the behavior of various substrates in the presence and absence of celecoxib. Steroids with non aromatic ring A seem to be among the bes t active substrates of SULT2A1 as evidenced by the values for their activities in the control tubes, with no celecoxib present. The order of sulfonating efficiency of these compounds is Epi isomer Epi T seems to be 2.5 ti OH E2 and E2 is twice as active as a SULT2A1 substrate E2. Considered in conjunction with previous report that the Kcat/K m values for the Z hydroxytamoxifen were higher than those for the E enantiomers (Apak and Duffel, 2004) these fin d ings throw more light on the isomer/enantiomer selectivity of SULT2A1. The molecule that was sulfonated to any significant extent apart from the estradiol analogues and the non aromatic steroids was E1. This was expected as it has a
77 structure very similar E2 viz. 6D E2, 9D E2, Eqn, Eq and 3Me E2 in the absence of celecoxib. A look at their docked structures suggests that this could be due to their orientations being close to neither the catalytic no r the non catalytic conformations of the crystallized DHEA. In the presence of celecoxib the flatter molecules that could slide into the E 2 in 3D structure, i.e. those with a double bond conjugated to the aromatic ring 6D E2, Eqn and 9D E2 showed the highest stimulation of overall sulfonation. The increase in the sulfonation was entirely due to the facilitation of 17 sulfonation by celecoxi b modul ation as the pres ence of celecoxib inhibited the 3 sulfonation. Absence of the conjugated double bond and the consequential twist in the molecular shape seems to have hindered the stimulation of Eq sulfonation to similar extent. In the case of 3Me E 2, prior celecoxib bonding should be eliminating unproductive binding of the substrate in the binding site as this is one compound along with Eqn which showed no signs of plateauing of the 17 sulfonation within the celecoxib concentration range tested (0 8 0 M). The sulfonation of non aromatic steroids tested viz. DHEA, AD, Epi T and T were inhibited by the celecoxib binding as these molecules are not flat enough to reach the His 99 for catalysis in the vertical conformations. It is interesting that inhibit ion of th ese compound s by celecoxib was not very potent. This may be due to the difficulty of replacing these compounds from the active site: they all have good affinities to the enzyme as evidenced by their control activities. So also the inhibition of 17 E2 due to
78 the tu rning away of the axial hydroxy group and that of E1 due to the inert keto group facing the critical His 99 were weak suggesting celecoxib is indeed replacing these substrates and binding in the substrate binding pocket. ST 60 in female rats with its Trp 16 inverted into the binding pocket allowed only 17 E2. To further the understand the differences between SULT2A1 and ST 60 and to appreciate the similarity of Trp 16 truncation of the binding pocket in ST 60 to celeco xib docking in SULT2A1 the sulfonation of non aromatic steroids can be tested with female rat liver cytosol. Interestingly, in the preliminary molecular modeling studies conducted, AD seems to be docking in ST 60 in a conformation that would allow 17 s ulf ation, which was not possible with SULT2A1. In summary, given the reports that celecoxib and its analogues have been found to have anti cancer effects in breast cancer (Lanza Jacoby et al., 2003; Basu et al., 2004; L evitt et al., 2004; Zhang et al., 2004) and colorectal cancer (Bertagnolli et al., 2006) both of which have estrogens as a component in the pathology, the effect celecoxib has on regulation of the availability of the unconjugated estrogens dem ands attention. The stimulation of 17 s ulfate generation can potentially be helpful in those conditions as this conjugate is resistant to sulfatase hydrolysis
79 Figure 4 1 E2 (magenta) docked in SULT2A1. The ligands are shown as sticks and the residues in the protein as wires. The enzyme colored by atom type, with oxygens in red, nitrogens in blue and carbons in brown. The amino acid residues in the binding site are labeled and their electrostatic surface is shown
80 Figure 4 2 Epi T (blue), T (green) docked in SULT2A1. The ligands are shown as sticks and the residues in the protein as wires. The enzyme colored by atom type, with ox ygens in red, nitrogens in blue and carbons in brown. The amino acid residues in the binding site are labeled and their electrostatic surface is shown.
81 Figure 4 3 6D E2 (magenta), 9D E2 (gold), Eqn (purple) and Eq (green) docked in SULT2A1. The l igands are shown as sticks and the residues in the protein as wires. The enzyme colored by atom type, with oxygens in red, nitrogens in blue and carbons in brown. The amino acid residues in the binding site are labeled and their electrostatic surface is sh own.
82 Figure 4 4 4OH E2 (cyan ), 2OH E2 ( light green) docked in SULT2A1. The ligands are shown as sticks and the residues in the protein as wires. The enzyme colored by atom type, with oxygens in red, nitrogens in blue and carbons in brown. The amino acid residues in the binding site are labeled and their electrostatic surface is shown.
83 Figure 4 5 E2 (green) and 3Me E2 ( salmon ) docked in SULT2A1. The ligands are shown as sticks and the residues in the protein as wires. T he enzyme colored by atom type, with oxygens in red, nitrogens in blue and carbons in brown. The amino acid residues in the binding site are labeled and their electrostatic surface is shown.
84 Figure 4 6 E2 an d celecoxib docked in SULT2A1. The ligands are shown as sticks and the residues in the protein as wires. The enzyme colored by atom type, with oxygens in red, nitrogens in blue and carbons in brown. The amino acid residues in the binding site are labeled a nd their electrostatic surface is shown.
85 Figure 4 7 E2 (dark blue), AD (light blue) and DHEA (green) docked in SULT2A1 in the presence of celecoxib. The carbons in celecoxib molecules are colored to match with the substrate. The ligands are shown as sticks and the residues in the protein as wires. The e nzyme colored by atom type, with oxygens in red, nitrogens in blue and carbons in brown. The amino acid residues in the binding site are labeled and their electrostatic surface is shown.
86 Figure 4 8 Epi T (blue), T (green) docked in SULT2A1 in the presence of celecoxib. The carbons in celecoxib molecules are colored to match with the substrate. The ligands are shown as sticks and the residues in the protein as wires. The enzyme colored by atom type, with oxygens in red, nitrogens in blue and carbons in brown. The amino acid residues in the binding site are labeled and their electrostatic surface is shown.
87 Figure 4 9 6D E2 (magenta), 9D E2 (gold), Eqn (purple) and Eq (green) docked in SULT2A1 in the presence of celecoxib. The ca rbons in celecoxib molecules are colored to match with the substrate. The ligands are shown as sticks and the residues in the protein as wires. The enzyme colored by atom type, with oxygens in red, nitrogens in blue and carbons in brown. The amino acid res idues in the binding site are labeled and their electrostatic surface is shown.
88 Figure 4 10 4OH E2 (cyan ), 2OH E2 ( light green) docked in SULT2A1 in the presence of celecoxib. The carbons in celecoxib molecules are colored to match with the s ubstrate. The ligands are shown as sticks and the residues in the protein as wires. The enzyme colored by atom type, with oxygens in red, nitrogens in blue and carbons in brown. The amino acid residues in the binding site are labeled and their electrostati c surface is shown.
89 Figure 4 1 1 E2 (green), 3Me_E2 (purple) and Eq (salmon) docked in SULT2A1 in the presence of celecoxib. The carbons in celecoxib molecules are colored to match with the substrate. The ligands are shown as stick s and the residues in the protein as wires. The enzyme colored by atom type, with oxygens in red, nitrogens in blue and carbons in brown. The amino acid residues in the binding site are labeled and their electrostatic surface is shown.
90 Figure 4 1 2 Comparison between human SULT2A1 and rat ST 60. The homology model of ST 60 is shown (in purple) along with human SULT2A1. Also shown E2 in ST 60 with its 17 hydroxy group in position for sulfonation. The ligands are shown as sticks and the residues in the SULT2A1 as wires. The enzyme colored by atom type, with oxygens in red, nitrogens in blue and carbons in brown. The amino acid residues in the binding site of SULT2A1 are labeled and their electrostatic surface is shown.
91 Fi gure 4 1 3 An overlap showing the binding of PAP or PAPS molecules in crystal structure s available in the Protein Data Bank. (PDB IDs represented are: 3U3J, 3U3K, 3U3M, 3U3O, 3F3Y, 3CKL, 3BFX, 2REO, 2Z5F, 2H8K, 2GWH, 2DO6, 2A3R, 1Q1Q, 1Q1Z, 1Q20, 1Q22, 1L S6, 1G3M and 1EFH). For all the structures oxygen atoms are shown in red, nitrogen atoms in blue and phosphorus atoms in orange.
92 Table 4 1 Activities (pmol/min/mg protein) of the substrates in the absence of celecoxib. In the case of the compou nds with both 3 and 17 Hydroxy group the activity with of the enzyme with respect to the major product (3 Sulfate for all of them except 17 E2) is given. The concentration of all the substrates is 400 nM. Sno A nalogue C ontrol activity 1 Epi T 1100 2 AD 1125 3 DHEA 1050 4 T 400 5 17 E2 275 6 E1 110 7 9D E2 16 8 6D E2 51 9 3Me E2 12 10 17 E2 130 11 Eqn 56 12 Eq 46 13 14 4OH E2 2OH E2 15 2
93 CHAPTER 5 TRICLOSAN A S THE SUBSTRATE AND INHIBITOR OF CYTOSOL IC SULFOTRANSFERASES Introduction tric hloro hydroxydiphenyl ether, shown in Figure 5 1) is a chlorinated phenolic compound used for its bactericidal activity as the active ingredient in various cosmetic products such as antiseptic soaps, tooth pastes, fab rics and plastics. It has been on the market as an ingredient in deodorants since 1960s and has been introduced in surgical scrub s at 1% in 1972 (Jones et al., 2000) It is regulated by the FDA as an over the counter drug for use in personal care products such as toothpastes and soaps and by EPA as an anti microbial agent in polymers and plastics (Fang et al., 2010) and a boilin (Dann and Hontela, 2011) It was found to (Bhargava and Leonard, 1996) Triclosan has been found in the raw sewage in concentrations 0.02 2.7 g/m L and is persistent as a micropollutant in wastewater treat ment effluents of various countries such as the USA, Australia, Germany, Spain, Sweden, Switzerland and the UK. (von der Ohe et al., 2012) Triclosan is highly lipophilic and can be absorbed by humans via mucous membranes of the buccal cavity and t he gastrointestinal tract after swallowing of personal care products such as dentifrices and mouthwashes or can penetrate skin upon topical application as deodorant sprays and soaps and is eliminated mainly by urine. Triclosan was reported to be present in human milk in Swedish (<20 to 300 g per kg lipid in 3 out of 5 samples tested) and Amercian mothers (from 100 to 2100 g
94 per kg lipid in 52 out of 62 samples analyzed) (Adolfsson Erici et al., 2002; Dayan, 2007) The m etabolic fate of tric l osan upon administration through oral, buaccal cavity and percutaneous routes has been reported previously (DeSalva et al., 1989; Bagley and Lin, 2000; Lin, 2000; Moss et al., 2000; Sandbor gh Englund et al., 2006) DeSalva et al reported that both upon consumption of 2 mg dose of triclosan or brushing twice a day with toothpaste containing 2 mg of triclosan, almost all of the triclosan found in the blood by day 7 was conjugated to either g lucuronide or sulfate. Sandborgh Englund et al in contrast observed that after 2 6 hr of oral administration of 4 mg triclosan the plasma concentration of unconjugated triclosan was 30 35%. In a percutaneous absor p tion study, Moss et al reported that upon topical application of 64.5 mM triclosan, a small amount (6.3% of the total) was collected in the receptacle on the other side of the membrane as the unconjugated substrate or its glucuronide after 24 hr but about 23.7% of the total remained in the skin. Of the fraction remaining in the skin, the unconjugated substrate and its sulfate formed the majority until 4 hrs after administration, after which formation of triclosan glucuronide was observed. Triclosan is both a substrate and inhibitor of UDP glucuro nosyl transferases and sulfotransferases in human liver microsomes and cytosol respectively (Wang et al., 2004) The structural similarity of triclosan to estrogenic polychlorobiphenylols and thyroid hormones lead to the examination of its endocrine effects. It was found that triclosan in hibits the sheep placental estrogen sulfotransferase mediated sulfonation of estradiol in a mixed competitive/uncompetitive way with a K ic of 0.09 0.01 nM and a K iu of 5.2 2.9 nM. It also inhibited estrone sulfonation with an IC 50 value of 0.6 0.06
95 nM (James et al., 2010) In another study tric l osan was shown to be capable of displacing estradiol from estrogen receptors of MCF7 cells and also from recombinant rs and increasing the growth of MCF7 cells over 21 days. The same study showed that triclosan can be anti androgenic also Triclosan at 0.1 M and 1 M concentrations could inhibit the induction of androgen responsive LTR CAT reporter gene in S115 mouse ma mmary tumor cells and in T47D human breast cancer cells by 1 nM and 10 nM testosterone respectively (Gee et al., 2008) In North American bull frogs, environmentally relevant levels of triclosan delayed thyroid hormone induced metamorphosis (Veldhoen et al., 2006) In both male and female weanling rats triclosan caused dose dependent decrease in serum thyroxine concentrations (Crofton et al., 2007; Zorrilla et al., 2009) Triclosan was also found to be an activator of pregnane X receptor, which i s involved in the transcription of en z ymes responsible for steroid and xenobiotic detoxification, with an efficiency of 53.8% of that of the positive control 10 M rifampicin (Jacobs et al., 2005) Of the Phase II enzyme systems responsible for the hitherto observed conjugation of triclosan, SULTs are expressed even in the human fetus (Barker et al., 19 94; Hume and Coughtrie, 1994; Hume et al., 1996; Richard et al., 2001; Stanley et al., 2001) Interference with the activities of these SULTs involved in steroid and thyroid hormone balance by triclosan can have detrimental effects on the fetal developmen t. The current study was designed to understand the sulfonation of triclosan, an important route of its biotransformation, by major sulfotransferase enzymes which are variously involved in sulfonations resulting in xenobiotic, thyroid, catecholamine and es trogen and other steroid biotransformation. The enzymes tested belong to two of the
96 major cytosolic sulfotransferase families SULT 1 (SULT1A1, 1A3, 1B1 and 1E1) and SULT 2 (SULT2A1). inhib its SULT2A1 and SULT1E1, the latter very potently (with an IC 50 value of 17nM E2 as substrate) It is also shown to inhibit cytosolic sulfotransferase activity in the human liver (Wang et al., 2004) Hence studies were also conducted to test the inhibitory activity of triclos an on other major SULTs viz. SULT1A1, 1B1 and 1A3. Also presented are the studies on the inhibition of triclosan on the sulfatase activity in sheep placenta. Materials and Methods Expression of R ecombinant S ulfotransferases All five sulfotransferase enzym es used in the study were expressed by the same method A bacterial expression system of XL 1 cells with pKK233 S vector subcloned with the SULT gene was used to express the enzyme as described previously (Falany et al., 1994). The glycerol stock was strea ked on to an LB agar plate with 200 g/m L ampicillin and allowed to grow over night. A single colony from the plate was then introduced into a 50 m L LB broth with 200 g/m L ampicillin and was shaken over night at 37 C. This starter culture was used to inoculate 2 L LB with 200 g/m L ampicillin and C until the O.D 600 reached a value of 0.5 which took about 3 hrs. It was then induced with 0.5 mM IPTG and shaken for 4 hrs at room temperature. The cultur e was pelleted by centrifuging at 2500 g for 15 min and stored at 20 until next morning. The pellet was resuspended in lysis buffer pH 8 (0.75 M Tris Ultra pure, ICN; 0.25 M sucrose, Sigma 99+%; 0.25 mM EDTA, Sigma grade and 0.02 mg/m L lysozyme) and cool ed on ice for 1.5 hrs and stirred for 30 C to reduce the viscosity of the extract. It was centrifuged again and the pellet obtained was resuspended in 10 mM
97 TEA buffer (Sigma 99.5%), pH 7.4 containing 10% glycerol (Sigma, min. 99%), 1.5 mM DTT (Bi o Rad), 10 g/m L PMSF (Fluka) and sonicated in four 10 sec bursts with 30 sec cooling in ice in between. The suspension was then ultra centrifuged at 100,000 g to obtain the cytosol as the sup ernatant which was saved at C for further analysis. The pr otein content of the crude extracts was determined by Bio Rad micro assay for proteins utilizing bovine serum album (2 10 g/m L ) standard curve. SDS PAGE was performed on 12% Tris HCl gels using Tris/Glycine/SDS running buffer and biosafe C oomassie stai ning reagent purchased from Bio Rad. The SULT content of each extract was estimated by using ImageJ software and used for the enzyme activity calculations. All of the plots shown and the enzyme kinetic data presented were generated in GraphPad Prism 4 soft ware Triclosan as a S ubstrate Appropriate volumes of concentrated triclosan stock solutions in ethanol were added to the reaction tubes and the solvent was evaporated before adding 10 mM Tris Cl, pH 7.4, 5 mM MgCl 2 2% BSA (BSA was not added to SULTs 1A3 and 2A1 reactions) along with 0.025 to 1.4 g of SULT and adjusted to 100 L final volume with water (0.025 g SULT1A1, 0.04 g SULT1A3, 0.035 g SULT1B1, 0.19 g SULT1E1 and 1.4 g SULT2A1). The amounts of enzymes used were adjusted to use less than 20% o f the triclosan in the kinetic studies and l ess tha n 30% of the co factor PAPS in the reaction for min before the reactions were started with 2 M 35S PAPS and were incubated for 10 min more and then stopped with 0.1 m L 1:1 sol ution of 2% acetic acid and tetrabutylammonium sulfate and 0.3 m L water for all of them except SULT1A1 Triclosan sulfate was separated from the substrate by solvent extraction of the reactions
98 by adding 2 m L of wa ter saturated ethyl acetate to the mixtures. The solvent mixture was vortex mixed and centrifuged to separate phases. Extraction was repeated two more times with 1 m L water saturated ethyl acetate each time. All the ethyl acetate fractions were combined in a vial and the solvent was evaporated before the scintillations were counted in a Beckman LS 6500 multipurpose scintillation counter. For SULT1A1 experiments, the reaction was stopped with 0.1 m L methanol and the 25 L of the mixture was spotted on microc rystalline cellulose (Avicel) thin layer plates and developed with a 3:1:1 mixture of n butanol, acetic acid and water as the mobile phase Various concentrations of triclosan and quercetin were tested for their inhibition of sulfonation with SULTs 1A1, 1A 3 and 1B1 using p NP, Dopamine and T3 as acceptor substrates respectively. The 100 L final volume of these reaction mixtures contained 50 mM Tris Cl, 5 mM MgCl 2 with 2% BSA (except for dopamine sulfonation) along with 4 M p NP in the case of SULT1A1, 10 M dopamine in the case of SULT1A3 and 100 M T3 for SULT1B1 as substrates and DMSO solutions of triclosan or quercetin. The control tubes contained just DMSO, the v ehicle. For reactions with SULT 1A3 and SULT with 10 M 35S PAPS and incubated for 30 min more before being stopped by adding 100 L methanol. The reactions were then applied on TLC plates for analysis. For SULT1A3 experi ments with dopamine as the substrate, microcrystalline cellulose (Avicel) plates were used with a 3:1:1 mixture of n butanol, acetic acid and water as the solvent. For SULT1B1 experiments with T3 as the substrate silica gel plates with a florescent indica tor (Whatman LK5DF) were used with a 40:40:20:8 mixture of isopropanol, chloroform, methanol and water as the solvent as described previously
99 (Wang et al., 1998) for 1 min before starting the reaction with 20 M PAPS and incubated for 10 min more before they were stopped by adding 0.1 m L 3% tricloroacetic acid and 0.3 m L water. Relatively more hydrophobic p NP was separated from its sulfate by solvent extraction w ith methylene chloride 1 m L of water saturated methylene chloride was added to the mixtures, vortex mixed and the phases were separated by centrifugation for three times to collect the product in the aqueous phase. 0.02 m L of the aqueous solution was coun ted for scintillations in a Beckman LS 6500 multipurpose scintillation counter. Effect of Triclosan on Estradiol Sulfonation in the Presence of C elecoxib To study the effect of triclosan on the SULTs responsibl e for E2 sulfonation, a condition in which bot h the sulfates (3 and 17) are generated at quantifiable levels was needed contained 50 nM E2, 0.1 M Tris Cl (pH 7.4), 5 mM MgCl 2 human liver cytosol apart from 0 L reactions were incubated at 37 C f or 30 min before they were stopped by 0.3 m L methanol and analyzed by LC MS/MS as described above Triclosan I nhibition of Sheep Placental Sulfatase A ctivity Three different sheep liver microsomes were used as the source of sulfatase in the experiments to test the inhibitory effect of triclosan on hydrolysis. For one set of experiments, a non steroidal substrate 4 methylumbelliferysulfate at concentrations 0.02 to 0.4 mM was incubated with 0 to 50 M concentrations of triclosan with 5 g sheep microsomal protein and 0.05 M Tris Cl buffer, pH 7.0 in 0.5 m L final volume. The reaction was stopped by adding 2 m L methanol to the tubes. The protein in each reaction was allowed to flocculate for 10 min before it was centrifuged for 10 min at
100 2,000 rpm. The supern atants were analyzed for fluorescence immediately after adding 0.5 m L 1M Tris base. A standard curve was obtained by using solutions of 4 methylumbelliferone with concentrations from 0.1 M to 4 M. For the second set of experiments with 3 H estrone sulfat e as the substrate at 0.2 and 2 M concentrations the triclosan concentrations tested were 0 to 200 M and the amount of sheep microsomal protein used was 50 g. The tubes also contained 0.05 M Tris he reactions were stopped by adding 0.1 m L ice cold methanol and kept on ice for 20 min to flocculate the protein. The reactions were than centrifuged at 2000 rpm for 10 min to separate the insoluble protein precipitate from the supernatant s which w ere ana lyzed by HPLC. HPLC analysis of the reactions was conducted on a Beckman Gold Nouveau HPLC system equipped with UV and fluorescence detectors and an IN/US ram IN/US Systems Inc., Tampa, Florida). A C 18 reverse phase column (4.6 mm x 25 cm) with a C 18 pre column (Discovery system, Supelco, Bellefonte, PA) with a mobile phase at a constant flow rate of 1 m L /min with 0.005 M Pic A (tetrabutylammonium sulfate) in 60% methanol was used. Results Triclosan as a S ubstrate SULT1A1 generated sulfates of some components or impurities in the buffer and two of them were prominent enough to visualize on the TLC. Solvent extraction was not used to estimate SULT1A1 sulfonation of triclosan to avoid their interference and instead TLC was employed. Triclosan sulf ate spot on the TLC was ascertained by matching with that formed in the case of SULT1A3 inhibition experiment discussed below. A representative TLC of SULT1A1 sulfon a tion of triclosan experiment is shown
101 in Figure 5 2 Although the various SULT enzymes tes ted here showed different affinities and capacities as shown in Table 5 1, all of them were able to sulfonate triclosan. The phenol sulfotransferases had higher affinity towards triclosan than SULT 2A1, the hydroxysteroid sulfotransfe r ase. SULT 1A1 with a A pp K m value of 0.03 M had the highest affinity to triclosan (Figure 5 3 ) as ex pected, since it is the principal SULT in liver responsible for xenobiotic sulfonation. SULT 1A1 is followed in affinity by SULT 1B1 ( App K m = 0.04 M), SULT 1E1 ( App K m = 1 .2 M ), SULT 1A3 ( App K m = 7.69 M ) and SULT 2A1 ( App K m = 20.9 M) as shown in Figure 5 4 to Figure 5 7 That SULT 2A1 has the least affinity is to be expected as it prefers aliphatic hydroxy groups to the phenolic groups for sulfonation. Similarly SULT 1A3 whic h is a member of phenol sulfotransferase family and is commonly referred to as catechol amine sulfotransferase has higher affinity to substrates with hydrophilic group attached to their core. The absence of such hydrophilic groups on triclosan could explai n its lower affinity to SULT 1A3. In the case of SULT 1B1 which is also referred to as thyroid hormone sulftransferase, the relatively low App K m and high App V max values for triclosan sulfonation could be explained by the similarity between its structure an endogenous substrates, triiodothyronine (T3) and tetraiodothyroxine (T4) (Figure 5 1 ) All the SULTs tested showed marked substrate inhibition with increasing concentrations of triclosan They exhibited two different forms of su bstrate inhibiti on patterns In the case of SULTs 1E1, 1A3 and 2A1 the rate of the reaction measured in nmoles of product formed by 1 mg of enzyme in 1 min decreased with increasing concentration of triclosan after reaching the maximum rate and eventually tended towards zero. This is the regular model of substrate inhibition kinetics and follows the
102 equation: Y=V max *X/[K m + X*(1+X/K i )], where Y is the rate of reaction, X the concentration of the substrate and Ki is the inhibitory concentration. For SULTs 1A 1 and 1B1 the rate did not converge on to the X axis but stabilized in to a plateau which is well below the maximum rate. This type of partial substrate inhibition was reported in human estrogen sulfotransferase with estradiol as the substrate previously (Zhang et al., 1998) and it follows the equation: Y= V 1 (1+(V 2 *X/V 1 *Ki ) )/(1+K m /X+X/K i ) where, V 1 is the maximum rate (could be considered V max ) and V 2 is the rate of reaction in the plateau phase of the rea ction and this velocity is achieved when all the active sites of the enzyme are saturated with one molecule of substrate and are in dynamic equilibrium with the second substrate molecule (Figure 5 8 ) The App V max and App K m values used for both equations we re obtained from linear portion of the kinetic curves for each enzyme tested. Triclosan I nhibition of S ulfotransferase A ctivities The activities of all three SULTs towards their substrates were inhibited by triclosan in a concentra tion dependent manner S ULT 1A1 (Figure 5 9 ) and 1B1 (Figure 5 10 ) were inhibited more potently with IC 50 values of 3.6 and 2.1 M respectively than SULT 1A3 (IC 50 = 31.1 M, Figure 5 1 1 ) The TLC separation of the mixtures in the case of dopamine and T3 sulfonation experiments s howed that both SULT 1A3 and SULT 1B1 also catalyzed triclosan sulfonation (Figure 5 1 2 and Figure 5 1 3 ) Effect of T riclosan on E stradiol S ulfonation in the P resence of C elecoxib les) showed about 25% substrate conversion and was chosen for the experiments with the inhibitors. The presence of celecoxib in the reaction mixtures caused the amount of 17 E2 17S formed in the control tubes to be about 50% of that of 17 E2 3S.
103 Triclos an (Figure 5 1 4 ) decreased amount of 17 E2 3S generated in a concentration dependent manner with an IC 50 value of 1 M The amount of 17 E2 17S generated on the other hand, remained unchanged for the concentration of triclosan required for reaching the I C 50 values for their 17 E2 3S inhibition. Triclosan I nhibition of S heep P lacental S ulfatase A ctivity Triclosan potently inhibited the hydrolysis of 4 methylumbelliferysulfate Figure 5 1 5 shows the kinetics of sulfatase at different substrate and inhibito r concentrations. The 1/V max plotted against triclosan concentration is a in the range tested (0 20 M) With all the five concentrations of the substrates from 0.02 to 0.4 mM the IC 50 values were between 7.5 to 18.6 M for the three sheep tested (Table 5 2 ). Statistical analysis by a two way anova showed no significant differences between the individual values obtained within 95% CI. This independence of the concentration of substrate suggests that triclosan is a non competitive inhibitor of 4 methylumbell ifery l sulfate hydrolysis in sheep placental microsomes. Triclosan inhibited the sulfatase activity towards estrone sulfate only at high micro 50 values for three different sheep placental 50 respectively. Representative inhibition curves for both substrate concentrations are given in Figure 5 1 6 Discussion Triclosan was shown to inhibit sulfonation of 3 hydroxybenzo(a)pyrene, p n itrophenol and acetaminophen in human liver cytosol previously (Wang et al., 2004) In sheep placental microsomes it inhibited the sulfonation of estradiol and estrone in low
104 and sub nano molar range (James et al., 2010) A catalytic profile of triclosan with major human sulfotransferases, which are involved in steroid, catecholamine and thyroid hormone regulation and xenobiotic detoxification w as necessary to understand the possible impact of the environmental presence of triclosan. Among th e five major SULTs studied by here SULT 1A1 showed highest affinity ( App K m = 0 .03 M) towards triclosan. SULT 1A1 is the most abundant SULT in human liver (Ric hes et al., 2009) and is the major xenobiotic metabolizing form and hence this ac tivity was to be expected. SULT 1B1, which constitutes 31% of total SULT mRNA in kidney and is the most abundant SULT in small intestine (36% of total SULT mRNA) was found achi eve a high rate for sulfonation of triclosan ( 31.2 nmole/min/mg enzyme) with an affinity ( App K m = 0.04 M) more similar to t hat with SULT1A1 and SULT 1E1 than with SULT1A3 and SULT2A1. The structural resemblance of triclosan with T3 and T4 the natural subs trates for SULT1B1 could explain why it is a good substrate for SULT1B1 Diiodothyronine (T2) sulfonation in rat liver cytoso l with an IC 50 value of 3.1 M (Schuur et al., 1998) SULT 1E1 has a moderate affinity ( App K m = 1 .2 M) to triclosan but the App V max (7. 6 nmole/min/mg enzyme) is low compared to 1B1. It has been shown previously that triclosan inhibits the sulfonation of 3 OH BaP with recombinant SULTs 1A1, 1B1 and 1E 1 in a concentration dependent manner (Wang et al., 2004) and the fact that triclosan is also a substrate to all these SULTs partly explains the inhibition. Triclosan was also sulfonated by SULT 1A3 and SULT 2A1 but with less affinity ( App K m values, 7.69 M and 20.9 M respectively). SULT 1A 3 is the major enzyme for catecholamine sulfonation and it has been shown that the residues Glu 146 and Glu 89 in its catalytic site make it prefer
105 substrates with hydrophilic groups, such as the amine group in dopamine. Triclosan has no such moieties and h ence shows lower affinity. SULT 2A1 is the hydroxysteroid sulfotransfease and its prefe rence to compounds with hydroxy groups precludes higher affinity towards triclosan. Substrate inhibition has been observed before in major SULT forms with various subst rates (Zhang et al., 1998; Rehse et al., 2002; Gamage et al., 2003; Gamage et al., 2005) and it can occur by more than one mechanism. In one mechanism, binding of a second substrate (p NP) molecule in the active sit e of the enzyme (SULT1A1) in a non productive orientation simultaneously was found to be inhibiting the activity (Gamage et al., 2003) In another study, the binding of the substrate (DHEA) in a non catalytic mode, which precludes the catalytic binding in the active site (of SULT2A1) was found to be resulting in substrate inhibition (Rehse et al., 2002) A third mechanism was also E2) goes further into the active site (of SULT1A1) than needed for achieving the hydrogen bond with catalytic His 108 (Gamage et al., 2005) p hosphate of PAP. This binding renders the enzyme complex inactive as it prevents PAP from leaving the co factor binding site. Triclosan exhibited substrate inhibition with all the SULTs tested. SULT1 A 3, 1E1 and 2A1 followed regular substrate inhibition kinetics whereas SULT1A1 and 1B1 followed kinetics described previously (Zhang et al., 1998) Triclosan inhibition of individual SULT activity towards their typical substrates and the inhibition of E2 3S generation in human liver cytosol agree with the previous observation that it inhibits sulfotransfer ase activity (Wang et al., 2004) The fact that triclosan is a substrate for all the major SULTs and the previous reports of its inhibition
106 of sulfonation of endogenous and xenobiotic substrates of sulfotransferases warrants further study into the adverse effects of its environmental conta mination. Inhibition of SULT1A1, for instance, could compromise the potency of the liver to detoxify certain xenobiotics and the inhibition of SULT1B1 can cause thyroid hormone imbalance. The IC50 values for triclosan inhibition for SULT1A1, 1B1 and 1A3 wi th their typical substrates were higher than the App K m values as would be expected. But in the case of SULT1E1, the IC 50 E2 was 17 nM (unpublished work) which is significantly lower than the App K m value for triclo san as a substrate to the same enzyme (1.2 M). Although very interesting, this kind of inhibition is not new in the SULT context as such phenomenon was obser Ethyny l estradiol and SULT1A1 (Rohn et al., 2012) The IC 50 for triclosan in hibition of 1 E2 3S generation in the human liver cytosol is similar to that for SULT1A1 catalyzed p NP sulfation At the concentration of estradiol tested (50 nM) SULT1E1 undergoes substrate inhibition by more than 90% The unchanged E2 17S concentration within the range tested reflects the fact observed by our group previously that triclosan is not a potent inhibitor of SULT2A1 and provides evidence that among the SULTs expressed in human liver SULT1A1 is most likely to catalyze 3 S ulfonation at this concentration of 17 E2 Triclosan did not inhibit the activity of placental steroid sulfatase enzyme potently. The inhibition of hydrolysis with triclosan was more potent with 4 methylumbelliferysulfate as the substrate (IC 50 values from 7.5 to 18.6 M) than with estrone su lfate (IC 50 values >100 M) The enzyme source used was sheep placental microsomes which contain a mixture of other sulfatases along with the STS. Since
107 estrone sulfate is a specific substrate of STS it can be concluded that triclosan is not a very potent inhibitor of STS. In summary, triclosan was a substrate to all SULTs, with varying affinities and exhibited substrate inhibition with all of them. The descending order of the efficiency with which the SULTs catalyzed triclosan sulfonation is 1B1 > 1A1 > 1 E1 > 1A3 > 2A1. The sulfonation activity of all the three major SULTs towards their typical substrates was also inhibited by triclosan, SULT1B1 and 1A1 more potently than SULT1A3. Triclosan was less potent in inhibiting steroid sulfates when compared to su lfatases in general.
108 Figure 5 1. Structures of triclosan and thyroid hormones, triiodothyronine and thyroxine.
109 Figure 5 2 Representative thin layered chromatogram of SULT1A1 sulfonation of triclosan. The farthest tra velled spot (shown by the arrow) is triclosan sulfate and the concentration of triclosan in the figure increases from left to right from 0.075 to 0.5 M (n=2).
110 Figure 5 3. Sulfonation of triclosan (0 to 1000 M) by SULT1A1. The reaction mixtures (described in Materials and Methods) were incubated for 10 min at 37 C before being stopped by 0.1 ml methanol and applied on TLC plates for analysis. Michaelis M enten portion of the graph is shown in the inset (n=2).
111 Figure 5 4. Sulfonation of triclosan (0 to 1000 M) by SULT1B1. The reaction mixtures (described in Materials and Methods) were incubated for 10 min at 37 C before being stopped by 0.1 mL 1:1 solution of 2% acetic acid and PicA solution and the sulfates were extracted into ethyl acetate for analysis. Michael is M enten portion of the graph is shown in the inset (n=2).
112 Figure 5 5. Sulfonation of triclosan (0 to 2000 M) by SULT1E1. The reaction mixtures (described in Materials and Methods) were incubated for 10 mi n at 37 C before being stopped by 0.1 mL 1:1 solution of 2% acetic acid and PicA solution and the sulfates were extracted into ethyl acetate for analysis. Michaelis M enten portion of the graph is shown in the inset (n=2).
113 Figu re 5 6. Sulfonation of i triclosan (0 to 2000 M) by SULT1A3. The reaction mixtures (described in Materials and Methods) were incubated for 10 min at 37 C before being stopped by 0.1 mL 1:1 solution of 2% acetic acid and PicA solution and the sul fates were extracted into ethyl acetate for analysis. Michaelis M enten portion of the graph is shown in the inset (n=2).
114 Figure 5 7. Sulfonation of triclosan (0 to 1000 M) by SULT2A1. The reaction mixtures (described in Materials and Methods) were incubated for 10 min at 37 C before being stopped by 0.1 mL 1:1 solution of 2% acetic acid and PicA solution and the sulfates were extracted into ethyl acetate for analysis. Michaelis M enten portion of the graph is shown in the inset (n=2).
115 Fi gure 5 8 The mechanism of substrate inhibition observed with SULT1A1 and SULT1B1 with triclosan as the substrate. E is the sulfotransferase enzyme and its complexes with PAPS and triclosan (TCL) are shown as superscript and subscript of E, respectively. Triclosan sulfate (TCL S) and PAP formed as products are also shown.
116 Figure 5 9. Triclosan inhibition of SULT1A1 activity with 4 M [ 14 C] p n itrophenol (p Np) as the substrate. The reaction mixtures (described in Materials and Methods) were incubated for 10 min at 37 C before they were stopped by 0.1 mL 3% trichloroacetic acid and 0.3 ml water. p Np was separated from its sulfate by solvent extraction into methylene chloride and analyzed by scintillation counting (n=2).
117 Figure 5 10. Triclosan inhibition of SULT1B1 activity with 100 M triiodothyronine ( T3 ) as the substrate The reaction mixtures (described in Materials and Methods) were incubated for 30 min at 37 C before the y were stopped by adding 0.1 mL methanol and applied to TLC plates for analysis (n=2).
118 Figure 5 1 1. Triclosan inhibition of SULT1A3 activity with 10 M dopamine as the substrate The reaction mixtures (described in Mate rials and Methods) were incubated for 30 min at 37 C before they were stopped by adding 0.1 mL methanol and applied to TLC plates for analysis (n=2)
119 Figure 5 12 Autoradiogram the reaction mixtures measuring triclosan inhibition of dopamine sulfonation. Dopamine sulfate is indicated by the arr ow and the bottom most bands correspond to PAPS. Independent experiments generating triclosan sulfate by SULT2A1 catalysis (data not shown) confirmed the top most band to be triclosan sulfate. Other bands are unidentified sulfates generated by SULT1A3 (n=2 ).
120 Figure 5 13 Autoradiogram the reaction mixtures measuring triclosan inhibition of T3 sulfonation. T3 sulfate is indicated by the arrow and the bottom most bands correspond to PAPS. Other bands are unidentified sulfates generated by SULT1 B1 (n=2).
121 (A ) (B) Figure 5 1 4 Triclosan inhibition of E2 3S generation by pooled human liver cytosol. (A) Histogram of activity vs. Triclosan concentration. Two individu al experiments were conducted for every triclosan concentration and each of these reactions was analyzed by LC MS/MS in triplicate. Data points in the plots are Mean SD for all six measurements. (B) Dose response curve for triclosan inhibition (control w ith no triclosan present is taken as 100 and others are plotted as percent of control).
122 (A) (B) Figure 5 1 5 Triclosan inhibition of 4 Methylumbelliferyl sulfate hydrolysis by sulfata se enzyme. (A) Michaelis M enten kinetics of inhibition with substrate concentrations 0 0.4 mM by triclosan (0 20 M). (B) Plot of 1/V max vs. triclosan concentration.
123 (A) (B) Figure 5 1 6 Triclos an inhibition of estrone sulfate hydrolysis by steroid sulfatase in in control tubes with no triclosan (n=2 )
124 Table 5 1 Triclosan sulfonation by major human cytosolic sulfotransferases. The K m and V max values are calculated from the linear portions of the sulfonation curves. The values for Ki are obtained by using the K m and V max values in the equations gi ven in the results section. All plots and kinetic calculations were done using GraphPad Prism 4 software. SULT App K m App V max 1 App Ki App V max 2 Efficiency a (M) (nmol/min/mg) (M) (nmol/min/mg) (mL/min/mg) SULT1A1 0.03 9.6 0.4 8.8 320 SULT1A3 7.69 15.0 46.0 -1.95 SULT1B1 0.04 31.2 7.7 4.0 780 SULT1E1 1.20 7.6 16.3 -6.33 SULT2A1 20.90 10.8 49.0 -0.52 a The efficiency of sulfonation was calculated from the Michaelis Menten portion of each curve.
125 Table 5 2 The IC 50 ( M) values for triclosan inhibit i on of 4 methylumbelliferysulfate hydrolysis 4 MeUSO 4 (mM) Sheep1 Sheep2 Sheep3 0.02 18.6 13.0 13.7 0.05 16.1 15.4 10.1 0.1 0 13.4 11.1 8.3 0.2 0 10.7 8.1 10.3 0.4 0 13.2 8.0 7.5
126 CHAPTE R 6 CONC L USIONS Among the Phase II metabolic enzymes SULTs are second only to UDP glucuronosyltransferases (UGTs) in the volume of drugs they conjugate (Evans and Relling, 1999) SULTs, expressed in all m ajor organs of the human body, are mainly concerned with detoxification of xenobiotics and their elimination by kidneys. Their high affinity, compared to UGTs, and broad substrate specificity make them an important defense against xenobiotics in micro mola r range or lower. This high affinity is crucial in maintaining the hormonal balance as steroid and thyroid hormones are present in fairly low concentrations in the body. The sulfate forms of these hormones are more abundant, as evidenced by the very low ra tios of hormone to hormone sulfate concentrations in plasma and typically act as their transport forms. Less attention is paid to the Phase II enzymes than Phase I enzymes in general when it comes to assessing or predicting drug interactions. This is just ifiable, to an extent, when we consider that the studies on drugs metabolized by or that have interactions with Phase I enzymes, especially cytochrome P450s are numerous. But recent studies such as those showing a range of inhibitory potential s of NSAIDs t owards SULT1A1 (mefenamic acid, IC 50 = 3.7 M to naproxen IC 50 = 473 M) (Vietri et al., 2000) demand detailed studies on the action of such compounds on sulfonation. Studies reporting the modulatory action of a cox 2 selecti ve inhibitor celecoxib on SULT2A1 enzyme resulting in the switching of the major product concentrations with ethynylestradiol (Cui et al., 2004) E2 (Wang and James, 2005) suggest that these studie s need not be limited to non selective cox inhibitors.
127 The classes of compounds that can influence sulfonation are by no means limited to pharmaceuticals as it has been shown that dietary chemicals such as quercetin (Walle et al., 1995) and environmental chemicals such as hydroxyl ated polychlorobiphenyls (Wang and James, 2006; Wang and James, 2007) are potent inhibitors of various SULT s. Triclosan, an antibacterial ingredient of personal care products, plastics and fabrics is a persistent environmental chemical found around the world has also been shown to inhibit SULTs (Wang et al., 2004; James e t al., 2010) The main focus of this work was to assess the impact of two xenobiotics celecoxib and triclosan on the sulfonation pathway To achieve this, these compounds were tested for their activity on sulfatase enzyme, responsible for the hydrolysis of the sulfonated compounds, along with that on SULTs. Celecoxib did not inhibit sulfatases to great extent. Triclosan was a better inhibitor of membrane bound sulfatase s other than steroid sulfatase (STS) as it did not inhibit hydrolysis of estrone sulf ate, a specific substrate of STS, to the extent it inhibited the general sulfat a se substrate, 4 m ethylumbelliferyl sulfate. The studies of the celecoxib effect on SULT2A1 were designed to find the reasons behind the switching of product concentrations from substrates such as ethynylestradiol E2. After the initial findings that sheep and male rat could not be used as model animals for the celecoxib E2 17 E2 sulfonation the objective of finding the reason for the effect on human SULT2A1 was the focus of this research Estradiol analogue studies have shown that an aromatic 3 hydroxy group were essential for the switching to occur in the presence o f celecoxib. This observation
128 follows from the fact that AD with no aromatic 3 hydroxy group do not conform to the switching pattern observed in other analogues. All the analogues tested that have both the hydroxyl grou ps showed switching behavior. A plausible explanation for the differences in the switching patterns among those compounds and the limited inhibition of non aromatic compounds was sought by molecular modeling studies. The observation made previously (Yalcin et al., 2008) that celecoxib occupies the substrate binding pocket to exert its action was strengthened by the binding s observed here. The compoun E2 in their 3D structure, i.e. those with a flat aromatic A ring and double bonds in conjugation with this ring in ring B or C showed greater switching compared to those wit hout. The stimulation of overall sulfonation E2 and its ring B and C unsaturated analogues and catechol estorgens observed with increasing amounts of celecoxib can be attributed to two reasons, both of which can have an impact on the outcome. Firstly, the binding of celecoxib obviates the non catalytic binding of the substrate, a phenomenon that leads to substrate inhibition in SULTs in general. The second reason for the stimulation can be the favorable pi stacking interaction the phenylmethyl group of celecoxib has with the aromatic A rings of the compounds ex hibiting the switching and stimulation of sulfonation. The non aromatic steroids were not significantly inhibited by the celecoxib concentrations tested here owing possibly, to their higher affinities to SULT2A1 compared to other analogues and celecoxib itself, as evidenced by high control activities. These non aromatic steroids were shown to be less able to bind catalytically as they were not able to slide into the binding pocket truncated by celecoxib in silico
129 This observation strengthens the postulat ion that celecoxib competes with and replaces the substrates from their original binding conformations to modulate their sulfonation. The enzyme kinetic studies, additionally, help ed in enhancing the understanding of the SULT2A1 enzyme itself. SULT2A1 sulf onates the hydroxy groups of non aromatic steroids to a greater extent than the aromatic hydroxy groups on compounds such as E2. The comparison between the activities of isomeric compounds shows that the isomers, Epi isomers T E2, respectively. Also, DHEA and AD, both having a hydroxy group are sulfonated to a similar extent. The homology model of the rat ST 60, the enzyme analogous to SULT2A1, showed that most of the amino acids in the binding pocket were similar to those in SULT2A1. One crucial difference is that Gly 17 in human SULT2A1 is replaced by tryptophan in ST 60. It is plausible that the hydrophobic side chain of Trp 17 truncates the binding pocket much the same way as celecoxib does in SULT2A1 resulting in E2 17 sulfate generation. To further the understanding of SULT modulation to achieve generation of more E2 sulfonation, molecules similar to celecoxib can be examined. It has been proposed that a mechanism by which aspirin reduces carcinogenesis is by inhibiting SULT1A1 (Harris et al., 1998) The possible cause for arresting carcinogenesis was proposed to be the inhibition of mutagenesis brought about by the electrophilic free radicals generated by sulfate esters of N hydroxy aryl amines and N hydroxy heterocyclic amines found in certain drugs, cooked foods and tobacco smoke (DeBaun et al., 1970; Miller and Miller, 1981; Chou et al., 1995) Given the reports suggesting the
130 role of NSAIDs in inhibition of SULTs and carcinogenesis and the findings that celecoxib was effective in treatment and prevention of breast cancer in mouse model (Lanza Jacob y et al., 2003; Basu et al., 2004; Zhang et al., 2004) it will be interesting to find out the interactions of the NSAIDs and other cox 2 selective inhibitors with SULT2A1 an d possibly other SULTs. The enzyme kinetic studies with triclosan as a substrate and inhibitor of major SULTs in humans showed that SULT1A1 and SULT1B1 were not only the major enzymes responsible for triclosan sulfonation but were also inhibited by triclosan more potently than other SULTs, with the exception of SULT1E1. SULT1B1 and SU LT1A1 sulfonted triclosan with efficiency ( app V max / app K m ) values of 820 and 320 ml/min/mg. These two isoforms are the most abundant SULTs in small intestine and liver, respectively, and may contribute most to the first pass metabolism of triclosan at low e nvironmental levels The inhibition of these two enzymes in low micro molar range (IC 50 values of 2.1 and 3.6 for SULT1B1 and SULT1A1, respectively) inspires more questions about the mechanism of inhibition. The mechanism of triclosan inhibition of estradi ol in sheep placenta has been shown to be by mixed but predominantly competitive inhibition previously (James et al., 2010) A similar mechanism could be responsible for the inhibitio n of SULT1A1 and SULT1B1. The investigation of triclosan inhibition of SULT enzymes is essential to understand its pote ntial role as an environmental substrate that may be hazardous to human and animal health The mechanism of triclosan inhibition for indi vidual SULTs, mainly the three most inhibited ones viz SULT1A1, 1B1 and 1E1, can be understood
131 better in a study involving ligand docking experiments with the help of the available crystal structures of these enzymes.
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147 BIOGRAPHICAL SKETCH S riram Ambadapadi is the second of the three sons of A.B. Sastry and A.N. Dayavathi. He spent the major part of his childhood in Warangal, India and finished j unior college there with a concentration in biology, physics and chemistr y In July 2004 he graduated fr om Osmania University (Nalanda College of P harmacy, Nalgonda) with as a pharmacist with the An dhra Pradesh pharmacy council. Under the mentorship of Prof. Paul S. Braterman, he obtained his m a nalytical c hemistry at University of North Texas in May 2007. He started working towards his PhD in m edicinal c he mistry under the guidance of Prof. Margaret O. James at University of Florida in f all of 2007 and obtain ed his doctorate in December of 2012.