Effects of glucocorticoids on sulfotransferase 1a (SULT1A) activities and the efflux of sulfate conjugates

EFFECTS OF XENOBIOTICS ON SULFOTRANSFERASE 1A (SULT1A) ACTIVITIES AND THE EFFLUX OF SULFATED CONJUGATES SHERRY NGO YAN YAN NATIONAL UNIVERSITY OF SINGAPORE 2003 EFFECTS OF XENOBIOTICS ON SULFOTRANSFERASE 1A (SULT1A) ACTIVITIES AND THE EFFLUX OF SULFATED CONJUGATES SHERRY NGO YAN YAN (BSc Biochemistry, Massey University, New Zealand) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE IN BIOCHEMISTRY DEPARTMENT OF BIOCHEMISTRY 2003 Acknowledgement Many thanks to my fellow colleagues and labmates who have had to bear with my seemingly endless frustrations from all the unsuccessful experiments I had encountered in the course of completing this project. I am truly grateful for their endless support and encouragement they have given me throughout the course of my study. I also extend my sincere thanks to Prof Sit Kim Ping and Ms Lim Beng Gek for the Hep G2 cells, the use of the HPLC instrument and for providing guidance on certain technical aspects of my experiments. I also sincerely thank my supervisor, Dr Theresa Tan whom without, I would not have been able to successfully complete my project for this degree. I am grateful for all her guidance and advice she has given me throughout these past years. Last but not least, this project was made possible with Grant R183-000-059-213, which was funded by the National Medical Research Council (NMRC) of Singapore. Table of Contents Contents Page no. 1. Introduction 1.1 Drug metabolism 1.2 Sulfation 1.2.1 Sulfation: An Overview 1.2.2 PAPS Synthetase 1.2.3 Cytosolic Sulfotransferases 1.3 The Cytosolic SULT Superfamily 1.3.1 An Overview 1.3.2 SULT1 Family 1.3.3 SULT2 Family 1.4 Gene Expression And Regulation Of Cytosolic SULTs 1.5 Hepatic Vectorial Transport 1.5.1 An Overview 1.5.2 Hepatic Xenobiotic Uptake Transporters 1.5.3 Hepatic Xenobiotic Efflux Transporters 1.6 Effects Ff Glucocorticoids On Cytosolic SULTs And Xenobiotic Transporters 1 1 2 2 4 7 12 12 13 16 18 20 20 22 24 27 2. Objective And Scope Of This Work 29 3. Materials and Methods 3.1 Materials 3.2 Methods 3.2.1 Cell Culture Of Hep G2 3.2.2 Treatment Of Hep G2 With Glucocorticoids 3.2.3 Cell Viability 3.2.4 Assay of SULT1A1 and SULT1A3 Activities In Hep G2 3.2.5 Efflux Assays Of Sulfated Conjugates Of Dopamine And -Nitrophenol 3.2.6 Reverse-phase High-performance Liquid Chromatography (RP-HPLC) Detection and Separation Of The Sulfated Conjugates Of Dopamine And -Nitrophenol From Na235SO4 3.2.7 Assay Of PAPSS Activity (PAPS Generation Assay) 3.2.8 Statistical Analysis 3.2.9 RNA Isolation And Reverse-transcription (RT)-PCR Of SULT1A Isoforms And 30 30 31 31 31 33 33 34 35 36 36 37 Contents Page no. Xenobiotic Transporters 3.2.10 RT-PCR of SULT1A3 Followed By Chemiluminescence Detection 38 Results 4.1 Cell Counting And Cell Viability Of Hep G2 4.2 RP-HPLC Chromatograms From SULT1A Assays 4.3 SULT1A Assay: Time-Dependent Sulfation By SULT1A1 And SULT1A3 In Hep G2 4.4 SULT1A Assay: Effect Of DX And PN On SULT1A1 And SULT1A3 Activities 4.5 RT-PCR Detection: Effect Of DX And PN On SULT1A3 mRNA Expression 4.6 PAPS Generation Assay: Effect of DX and PN On SULT1A1 And SULT1A3 Activities 4.7 Efflux Assay: Effect of DX on Xenobiotic Transporters 4.8 RT-PCR Detection: Effect Of DX On Xenobiotic Transporters 4.9 Software Analysis: Putative Promoter Elements Of The SULT1A3 Gene 40 40 40 44 Discussion 5.1 Effects Of Glucocorticoids On Sulfation In Hep G2 Cells 5.2 DX Differentially Induces SULT1A3 But Not SULT1A1 Activity 5.3 Effects of DX On Efflux Of Sulfated Conjugates In Hep G2 Cells 5.4 Effects of DX On Detoxification Via Sulfation Pathway 68 68 6. Conclusion 75 7. Future works 76 8. References 78 9. Abbreviations 98 4. 5. 45 48 51 52 53 56 70 72 74 List of Figures Contents Figure 1.1 Figure 1.2 Figure 1.3 Figure 1.4 Figure 1.5 Figure 2.1 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7 Figure 4.8 Figure 4.9 Figure 4.10 Figure 4.11 Figure 4.12 Figure 4.13 Page no. Human PAPSS1 and PAPSS2 (next page) The highly conserved Region I and IV amino acid SULT signature sequences Proposed reaction mechanism of sulfuryl transfer catalyzed by SULTs The human SULT enzyme family Hepatic vectorial transport Schematic outline of the scope of this work Separation of -nitrophenyl-35sulfate from sodium-sulfate (Na235SO4) Separation of dopamine-35sulfate from Na235SO4 Separation of PAP35S from Na235SO4 Time-dependent (A) -nitrophenyl-ST and (B) dopamineST activities in Hep G2 (A) SULT1A1 and (B) SULT1A3 activities in Hep G2 following three days of DX treatment (A) SULT1A1 and (B) SULT1A3 activities in Hep G2 following three days of PN treatment (A) SULT1A1 and (B) SULT1A3 activities in preconditioned Hep G2 cells prior to three days of DX treatment (A) SULT1A1 and (B) SULT1A3 activities in preconditioned Hep G2 cells prior to three days of PN treatment RT-PCR of SULT1A3 and -actin in Hep G2 cells following three days of GC treatment Panel A: SULT1A3 mRNA levels in Hep G2 following three days of GC treatment PAPS generation by PAPSS in Hep G2 following three days of DX and PN treatment Efflux of (A) -nitrophenyl-35sulfate and (B) dopamine35 sulfate in Hep G2 following three days of DX treatment RT-PCR of various isoforms of MRP xenobiotic 5 9 12 16 21 29 41 42 43 44 45 46 47 47 48 49 51 52 54 Contents Figure 4.14 Figure 4.15 Figure 4.16 Figure 4.17 Figure 4.18 Figure 5.1 Figure 5.2 Page no. transporters from total RNA extract of DX-treated Hep G2 cells RT-PCR of various isoforms of OATP transporters from total RNA extract of DX-treated Hep G2 cells SULT1A3 cDNA sequence (GenBank Accession Number: U20499) Annotations of the human SULT1A3 genomic sequence (GenBank Accession Number: NT_042812) BLAST result from alignment of proximal 5’UTR of SULT1A3 cDNA onto the ~6.3 kb region proximal to the translational start site on SULT1A3 genomic sequence Putative regulatory factors and elements of the human SULT1A3 gene (A) Prednisolone and (B) Dexamethasone Potential response elements in the 5’-untranslated region of human MRP1 53 57 62 65 67 69 74 List of Tables Contents Table 1.1 Table 1.2 Table 1.3 Table 3.1 Table 3.2 Table 3.3 Table 4.1 Table 4.2 Table 4.3 Table 4.4 Page no. Phase II conjugation reactions Characteristics of human PAPSS1 and PAPSS2 Names of the corresponding SULTs that are listed in Figure 1.2, based on the new nomenclature and their GenBank Accession Numbers Buffer compositions of PBS and HBSS Solvent composition for the separation of dopamineand -nitrophenyl-sulfate from Na235SO4 Primer sequences of various isoforms of SULT 1A, MRP, OATP and the control, -actin for RT-PCR Typical cell concentration and viability of Hep G2 following three days of GC treatment Intensities of SULT1A3 blot following chemiluminescence detection of RT-PCR products mRNA levels of the various transporters in DX-treated Hep G2 cells Consenus sequences of SP1, AP1, AP2, CAAT and GRE used by MatInspector software 2 6 10 32 35 38 40 50 55 67 Summary Sulfation by sulfotransferases (SULTs) is pharmacologically important for detoxification of endogenous compounds and xenobiotics. Glucocorticoid (GC) regulatory elements have been identified for rat SULT1A1. In this study, the effects of dexamethasone (DX) and prednisolone (PN) on human SULT1A and 3’phosphoadenosine 5’-phosphosulfate synthetase (PAPSS) activities, and DX on mRNA expression of xenobiotic transporters were explored using Hep G2 cells. PAPSS activities were unaltered by both DX and PN. While SULT1A1 activity was unaltered by DX and PN, 10-7M DX increased SULT1A3 activity by 80% which correlated to the increase in mRNA levels of 1.8 folds. Software analysis of the 5’ flanking region of human SULT1A3 gene showed the presence of a consensus binding site for the GC receptor. Such a site was not present for SULT1A1. MRP and OATP isoforms were generally DX-inducible. MRP3 mRNA expression was down-regulated, whereas a biphasic response was observed for MRP2. Efflux of -nitrophenyl-sulfate was down-regulated by DX by nearly 50%; probably due to increased uptake, possibly by OATP proteins and/or reduced export. Dopamine-sulfate was up-regulated by 150% at 10-7 M DX; probably a result of increased efflux in addition to the increased SULT1A3 activity. 1. Introduction 1.1 Drug Metabolism Drug metabolism essentially comprises Phase I (functionalization reactions), Phase II (conjugative reactions) and Phase III (involving protein transporters for drug excretion). Phase I reactions generally include oxidation, reduction, hydrolysis, hydration although there exists other rarer reactions such as isomerization and dimerization, transamidation, decarboxylation, etc. (Kauffman, 1990). Phase II conjugations are carried out by a diverse group of enzymes acting on numerous types of compounds. The conjugation processes generally lead to bioinactivation of the drugs or xenobiotics to form water-soluble products that can be readily excreted through bile or urine. As such, Phase II reactions are said to be the true “detoxification” pathways since they generate the final inactive, excretable products of a drug or xenobiotic. Conjugation reactions that comprise the Phase II detoxification pathways, the enzymes involved and the types of drugs conjugated are as listed in Table 1.1 (Kaufman, 1990). Phase III transport systems will be discussed in Section 1.5. Conjugation reaction Enzyme Functional group Glucuronidation UDP-glucuronyltransferase -OH, -COOH, -NH2, -SH Glycosidation UDP-glycosyltransferase -OH, -COOH, -SH Sulfation Sulfotransferase -NH2, -SO2NH2, -OH Methylation Methyltransferase -OH, -NH2 Acetylation Acetyltransferase -NH2, -SO2NH2, -OH Amino acid conjugation Glutathione conjugation -COOH Glutathione-S-transferase Epoxide, Organic halide Fatty acid conjugation -OH Condensation Various Table 1.1 Phase II conjugation reactions (Kauffman, 1990) 1.2 Sulfation 1.2.1 Sulfation: An Overview Sulfation plays a role in homeostasis and regulation of many important endogenous chemicals such as catecholamines, steroids as well as other macromolecules (Coughtrie et al, 1998). In addition, it serves as one of the detoxification pathways for the various xenobiotics, although occasionally it results in the activation of the xenobiotic to a reactive electrophile (Buhl A et al, 1990; Falany, 1991; Glatt, 1997). The energy-requiring, sulfation process is catalysed by the substrate-specific sulfotransferases, using 3’-phosphoadenosine 5’-phosphosulfate (PAPS) and ATP as cosubstrates for the sulfation reaction. Sulfation reactions utilize PAPS as the sulfate donor. PAPS is made in the cytosol as a two-step enzymatic process (Robbins and Lipmann, 1958). Firstly, ATP sulfurylase catalyzes the formation of adenosine-5’phosphosulfate (APS) from inorganic SO4 in the presence of ATP. Subsequently, APS kinase catalyzes the formation of PAPS from the phosphorylation of APS (using ATP as the phosphate donor). The primary source of sulfur is free SO42-, which is transported into the cytosol by a variety of transporter or symporter molecules (Falany, 1997a; Falany, 1997b; Weinshilboum et al, 1997; Kullak-Ublick et al, 2000). For post-translational protein modification via sulfation, PAPS is delivered to the Golgi network with the aid of the PAPS translocase, where the secreted proteins can be sulfated by the substrate-specific membrane sulfotransferases (Mandon et al, 1994; Ozeran et al, 1996; Schwarrtz et al, 1998). For metabolism of endogenous compounds or detoxification of xenobiotics, the PAPS is utilized in the cytosol by the cytosolic sulfotransferases (Klassen et al, 1997). Sulfotransferases (SULTs) exist as cytosolic and membrane-bound enzymes. Cytosolic SULTs catalyze the sulfation of endogenous and exogenous small-molecule substrates like steroids, hormones, neurotransmitters and xenobiotics, including therapeutic drugs, in animals. In plants, similar reactions occur with flavonols (Coughtrie et al, 1998). Membrane-bound SULTs typically catalyze the sulfation of macromolecules, such as proteoglycans, glycosaminoglycans, polysaccharides and tyrosyl residues within proteins (Huttner, 1982; Hashimoto et al, 1992). 1.2.2 PAPS Synthetase ATP sulfurylase and APS kinase constitute the sulfate activation pathway in both higher and lower organisms. In simpler organisms (bacteria, yeasts, algae, protozoa), they exist as two separate and relatively small polypeptides (Klassen and Boles, 1997; Farooqui, 1980). However, in higher organisms including mammals, they exist as a single bifunctional enzyme, called the PAPS synthetase (PAPSS) (Lyle et al, 1994). Two different isoforms of PAPSS, PAPSS1 and PAPSS2, are known to exist in humans, mice and the marine worm Ureches caupo (Li et al, 1995; Rosenthal et al, 1995; Besset et al, 2000). The human PAPSS1 and PAPSS2 proteins show 76.5% amino acid sequence identity (Kurima et al, 1998; ul Haque et al, 1998). Historically, PAPS synthesis is assumed to occur exclusively in the cytosol. In fact, cytosol and Golgi apparatus are the only sites of PAPS utilization by known sulfotransferases (Falany CN, 1997a; Falany CN, 1997b; Bowman and Bertozzi, 1999). However, it has been reported that human PAPSS1 is a nuclear protein, in contrast to PAPSS2 which is cytosolic. Besset et al demonstrated that the APS kinase domain targets PAPSS1 to the nucleus in a number of mammalian cell lines (Besset et al, 2000). In addition, nuclear targeting of PAPSS1 in yeast functionally complements ATPsulfurylase and APS-kinase-deficient strains (Besset et al, 2000). Furthermore, ectopic PAPSS2 expression in mammalian cells dramatically localized the cytosolic PAPSS2 to the nucleus, when coexpressed with PAPSS1 (Xu et al, 2000). Human PAPSS1 and PAPSS2 are very similar in structure. Figure 1.1 shows that both genes are made up of 12 exons, but exon 1 (the first splice junction) contains an additional codon in PAPSS2. All exon-intron splice sites for the two genes are virtually identical. Introns of PAPSS1 vary from 1.6 kb to 21.9 kb whereas the introns of PAPSS2 are generally shorter than those of PAPSS1. Table 1.2 summarizes the characteristics of human PAPSS1 and PAPSS2 genes. The 5’-flanking region of PAPSS1 did not contain any TATA or CAAT sequences. The transcriptional start site did not contain an Initiator (Inr) sequence. However, a TATA box was located at 21 bp upstream of the transcription initiation site for PAPSS2 (Xu et al, 2000). Figure 1.1 Human PAPSS1 and PAPSS2 (Xu et al, 2000) PAPSS1 PAPSS2 GenBank Accession: AF097710-AF097721 Chromosome band 4q24 108 kb 12 exons 2.7 kb mRNA transcript No TATA box/motif GenBank Accession: AF160503-AF160509 Chromosome band 10q23-24 >37 kb 12 exons 4.2 kb mRNA transcript TATA motif at -21 bp from transcriptional start site Several putative Sp1 binding sites at 5’flanking region Slice junctions conform to ‘GT-AG’ rule Highly expressed in liver and adrenal gland Several putative Sp1 binding sites at 5’flanking region Slice junctions conform to ‘GT-AG’ rule Low expression in liver, skeletal muscle and adrenal gland Table 1.2 Characteristics of human PAPSS1 and PAPSS2 Schwartz et al showed that the rat PAPS synthetase uses a channeling mechanism to transfer APS from the sulfurylase to the kinase active site. The defect in PAPS production observed in brachymorphic mice was primarily due to the decreased ability to channel APS, hence, the inability to generate PAPS efficiently (Schwartz et al, 1998). Similar observations were made by Hastbacka et al and Lyle et al, who reported the brachymorphic mouse phenotype attributed to defects in the ATP sulfurylase/ APS kinase protein (Hastbacka et al, 1994; Lyle et al, 1995). More importantly, a variant sequence within the human PAPSS2 orthologue has been associated with spondiloepi-metaphyseal dysplasia, an inherited syndrome in humans, phenotypically similar to the brachymorphic phenotype in mice (ul Haque et al, 1998). This clearly signifies the role of PAPS synthetase in the generation of PAPS for sulfation. 1.2.3 Cytosolic Sulfotransferases The sulfotransferases (SULTs) constitute a diverse range of enzymes that make up an emerging superfamily. Historically, the reactions catalyzed by these low-capacity enzymes have been termed “sulfation”, although chemically, they are more accurately described as sulfonation. Sulfonation/sulfation by the sulfotransferases involves the transfer of sulfonate group from PAPS to the acceptor substrate (e.g. endogenous compound, neurotransmitter or xenobiotic) to form either a sulfate or sulfamate conjugate (Weinshilboum et al, 1997). Cytosolic sulfotransferases usually are found as hetero- and homodimers, with monomer molecular weight ranging from 30 to 36 kDa (Falany, 1991). However, in some plants and mammals, they can exist as catalytically active monomers (Takikawa et al, 1986). SULTs are single / globular proteins with characteristic five-stranded parallel -sheets. The -sheet constitutes the PAPS-binding site and the core active site of the enzyme. Consequently, both these sites are highly conserved in cytosolic and membranebound SULTs. As such, all sulfotransferases are categorized as members of a single gene superfamily. The membrane-bound SULTs are attached to the membranes of the Golgi network at the amino-terminal end (Negishi et al, 2001). Protein sequence alignments of cytosolic sulfotransferases of different species identified various regions of sequences that were highly conserved (Marsolais and Varin, 1995; Weinshilboum et al, 1997). As shown in Figure 1.2, two of those regions are located relatively near the termini of the protein sequence; one being near the amino terminus (Region I) and the other near the carboxy terminus (Region IV). Through the cloning of SULT cDNAs, the consensus sequence that has been identified in Region I is YPKSGTxW and in Region IV is RKGxxGDWKNxFT, where “x” represents any amino acid. The motif of Region IV is similar to the glycine-rich phosphate-binding loop (the “P-loop”), present in some ATP and GTP-binding proteins. Consequently, it is hypothesized that the portion of Region IV that contains the sequence GxxGxxK might be a homologue for the glycine-rich region, followed by a conserved lysine, present in some P-loop motifs (Komatsu et al, 1994). Through site-directed mutagenesis experiments in guinea pigs, the conserved G and K in this region were shown to be essential for enzymatic activity and the binding of 35S-PAPS as a photoaffinity ligand for the enzyme (Komatsu et al, 1994). Furthermore, similar studies with SULTs in plants had led to the conclusion that the invariant lysine within Region I might be important for the stabilization of an intermediate formed during the sulfonation reaction (Marsolais and Varin, 1995). Figure 1.2 (next page) The highly conserved Region I and IV amino acid SULT signature sequences (Weinshilboum et al, 1997) “Position” refers to amino acid number with the protein sequences for regions I and IV. “x” represents any amino acid. Black columns denote amino acids with > 93% identity with residues at that position. Black columns with an asterisk denote > 93% identity within groups of amino acids having similar polarity. White boxes represent non-identical residue. Arrows indicate invariant amino acids. 9 9 The enzyme names used in Figure 1.2 were cDNA names based on the old nomenclature. The new nomenclature for the corresponding SULT cDNA/enzymes is as listed in Table 1.3. Old Name rPST mPST hTSPST1 hTSPST2 mfPST hTLPST bPST r1B1ST rAAFST bEST hEST gpEST mEST rEST rEST-6 rSMP2 rHSST1 rHSST2 rHSST3 mfHSST hDHEAST gpHSST1 gpHSST2 fcFST3 fbFST3 fcFST4 atST Table 1.3 New Name Rat SULT1A1 Mouse SULT1A1 Human SULT1A1 Human SULT1A2 Monkey SULT1A1 Human SULT1A3 Bovine SULT1A1 Rat SULT1B1 Rat SULT1C1 Bovine SULT1E1 Human SULT1E1 Guinea pig SULT1E1 Mouse SULT1E1 Rat SULT1E1 Rat SULT1E1 Rat SULT2A1 Rat SULT2A1 Rat SULT2B1 Rat SULT2B1 Monkey SULT2A1 Human SULT2A1 Guinea pig SULT2A1 Guinea pig SULT2B1 Flaveria chloraefolia (plant) SULT-like flavonol Flaveria bidentis (plant) SULT-like flavonol Flaveria chloraefolia (plant) SULT-like flavonol Arabidopsis thaliana (plant) SULT-like flavonol Accession No. X52883 L02331 L19999 X78282 D85514 L19956 U35253 U38419 L22339 X56395 U08098 U09552 S78182 S76489 S76490 J02643 M31363 M33329 D14989 D85521 U08024 U06871 U35115 M84135 U10275 M84136 Z46823 Names of the corresponding SULTs that are listed in Figure 1.2, based on the new nomenclature and their GenBank Accession Numbers SULTs were traditionally named after the substrates they catalyze. However, this form of naming system is often misleading and confusing because different SULTs show overlapping substrate specificities. As such, a systematic nomenclature, similar to that used for classifying the cytochrome P450 enzymes, is in use but not yet finalized for the SULTs. For this new nomenclature, members of each family is indicated by a number after “SULT”; and members of each subfamily is indicated by a letter after each subfamily number. More recently, using the human estrogen SULT (hEST) which is responsible for sulfation of estrogens, Negishi et al demonstrated that the conserved lysine (K47) within Region I and another highly conserved serine (S137 in hEST or S138 in mouse EST) are essential not only for PAPS-binding site, but also for catalysis. Figure 1.3 shows the sidechain nitrogen of the conserved lysine forms an H-bond with an O-atom of the 5’phosphate group in the PAPS molecule. The hydroxyl side-chain of the conserved serine interacts with an O-atom in the 3’-phosphate. X-ray crystallography of hEST also showed that the side-chain of the conserved Ser137 interacted with the side-chain of the conserved K47. As a result of the interaction, the side-chain nitrogen of the conserved lysine is repelled from the bridging oxygen of the PAPS molecule. It was also observed that the serine decreased PAPS hydrolysis when the substrate was absent from the active site. However, mutation of the serine residue markedly increased PAPS hydrolysis. This led to the conclusion that the conserved serine may serve to regulate the sulfuryl transfer process by interacting with the catalytic lysine (Negishi et al, 2001). In addition, using x-ray crystals of mouse EST (mEST)-PAP-vanadate, Negishi et al also demonstrated that the conserved histidine at position 108 (H107 in human) and the conserved lysine at position 48 (K47 in human) appeared to be catalytic residues. He reported that mutation of His107 of the hEST to asparagine rendered the enzyme incapable of hydrolyzing PAPS nor catalyzing the sulfation reaction (Negishi et al, 2001). Figure 1.3 Proposed reaction mechanism of sulfuryl transfer catalyzed by SULTs (Negishi et al, 2001) Residue numbers are taken from hEST. 1.3 The Cytosolic SULT Superfamily 1.3.1 An Overview Presently, at least 44 cytosolic sulfotransferases have been identified in mammals, ranging from rats and mice to dogs, rabbits, cows, guinea pigs and monkeys. In humans, at least 11 different sulfotransferases have been identified (Nagata and Yamazoe, 2000). These enzymes are classified into three sub-families based on their amino acid sequence identity and substrate specificity (Yamazoe et al, 1994; Weinshilboum et al, 1997). Members of the sub-family SULT1 preferentially sulfate phenols (including estrogens and iodothyronines) and catechols (including catecholamines). SULT2 family members mainly sulfate steroids, sterols and other alcohols (Yamazoe et al, 1994; Strott, 1996; Weinshilboum et al, 1997). In general, amino acid sequence comparisons between members of each family yield at least 45% similarity. However, members of subfamilies show at least 60% amino acid sequence identity (Nagata and Yamazoe, 2000). 1.3.2 SULT1 Family As shown in Figure 1.4, the human SULT1 family, presently known to be the largest family, comprises of SULT1A, SULT1B, SULT1C and SULT1E enzymes. The human SULT1A subfamily has three members namely, SULT1A1, SULT1A2 and SULT1A3. These three genes differ by less than 10% at amino acid level (Figure 1.4) and are physically mapped to a small chromosomal region 16p. SULT1A1 preferentially sulfates simple phenols. Classical phenolic substrates are -nitrophenol and -napthol, although weaker activities have been observed with sulfation of catechols, hydroxyarylamines, and iodothyronines. SULT1A1 also sulfates the common xenobiotics, acetaminophen and minoxidil (Reiter et al,1982; Young et al, 1988; Falany and Kerl, 1990; Duanmu et al, 2000; Honma et al, 2001). SULT1A2 sulfates simple phenols and catechols, albeit at a lower catalytic activity and shows a higher Km value when compared to SULT1A1 (Dooley, 1998a). SULT1A3 has been observed to preferentially catalyze the sulfation of catecholamines; the classical substrate being dopamine. It has only a limited activity for -nitrophenol (Veronese et al, 1994; Honma et al, 2001). Other substrates for SULT1A3 include tyramine, 5hydroxytryptamine, salbutamol, isoprenaline and dobutamine (Honma et al, 2001). Human SULT1A1 and SULT1A2 are mapped to the chromosomal position 16p12.1-p11.2 and are approximately 45 kb apart (Her, 1996; Gaedigk, 1997). SULT1A3 is localized 100 kb away (Dooley, 1998b). SULT1A1 is expressed in many tissues but is abundant in the human liver, brain, kidney and platelets. However, SULT1A2 apparently is expressed only in the adult human liver as well as in the fetal liver and spleen. SULT1A3 is also expressed in the fetal liver and brain, with lower levels in the lung and kidney (Dooley et al, 2000; Nagata and Yamazoe, 2000). To date, only one form of SULT1B (SULT1B1) has been identified in human. SULT1B1, localized on chromosome band 4q13, is highly expressed in the liver (Dooley et al, 2000; Meinl and Glatt, 2001). Although not much is known about this isoform, SULT1B1 has been shown to catalyze sulfation of 3,3’,5’-triiodothyronine and nitrophenol in human and rat livers, at a much lower affinity. These substrates are sulfated by members of the SULT1A family at a much higher affinity (Dunn et al, 2000; Tsoi et al, 2001). SULT1C was first isolated from rat as an N-hydroxy-2-acetylaminofluorenespecific sulfotransferase (Nagata et al, 2000). Since then, two human SULTs have since been identified through the EST database, namely SULT1C1 and SULT1C2 (Her et al, 1997). Although not much is known about these enzymes presently, SULT1C1 and SULT1C2 share about 63% identical at the amino acid level as shown in Figure 1.4. SULT1C2 is thought to be a “dead” enzyme because it shows little or no activity towards any standard substrates; probably due to an amino acid change in the active site of the enzyme (Coughtrie and Johnston, 2001). SULT1C is localized to the human chromosome band 2q11.1-11.2 (Her et al, 1997). Only one form of human SULT1E has been identified so far, and it is named SULT1E1. SULT1E1, found on chromosome band 4q13.1, is known as a typical estrogen SULT, with the Km value for -estradiol being the lowest among the human SULTs. Experimental data suggests that estrogen sulfation is the main physiological role of SULT1E1 in humans (Nagata and Yamazoe, 2000). SULT 1A1 SULT 1A2 SULT 1A3 SULT 1B1 SULT 1C1 SULT 1C2 SULT 1E1 SULT 2A1 SULT 2B1a SULT 2B1b SULT 4A1 SULT 1A1 . SULT 1A2 SULT 1A3 SULT 1B1 SULT 1C1 SULT 1C2 SULT 1E1 SULT 2A1 SULT 2B1a SULT 2B1b SULT 4A1 96 93 53 53 51 50 34 33 33 32 . 90 57 53 51 49 34 33 33 32 . 52 53 50 48 34 34 34 34 . 53 52 54 36 34 34 32 . 62 48 34 31 31 33 . 47 35 32 32 31 . 34 32 32 33 . 48 43 27 . 97 29 . 29 . Figure 1.4 The human SULT enzyme family (Weinshilboum et al, 1997) Amino acid similarities between members of the SULT superfamily. SULT4 represents a novel SULT which has yet to be characterized. 1.3.3 SULT2 Family Relative to the SULT1 family, limited studies have been done with the characterization of members of the SULT2 family. Currently, SULT2 family comprises SULT2A and SULT2B. More than one form of SULT2A have been isolated from rodents and they exhibit different substrate specificity on the sulfation of hydroxysteroids (Homma et al, 1996; Yoshinari et al, 1998). However, the single form expressed in humans, hSULT2A1 catalyzes the sulfation of hydroxysteroids, including bile acids (Otterness et al, 1995). As such SULT2A1 is also known as dehydroepiandrosterone sulfotransferase (DHEA-ST), DHEA being its prototypic hydroxysteroidal substrate. Located on chromosome band 19q13.3, the human SULT2A1 consists of 6 exons and is highly expressed in the adrenals, liver and the intestine (Durocher et al, 1995; Luu-The et al, 1995). Figure 1.4 also shows that human SULT2B1 is 48% homologous to SULT2A1 at the amino acid level and is mapped to the chromosome band 19q13.3, approximately 500 kb telomeric to the location of SULT2A1 (Her et al, 1998; Nagata and Yamazoe, 2000). In human, it is reported that the single gene of SULT2B1 encodes two different forms of this enzyme, SULT2B1a and SULT2B1b. Also known as hydroxysteroid SULTs, SULT2B1a and SULT2B1b were demonstrated to catalyze the sulfation of the prototypic hydroxysteroid SULT substrate, DHEA (Her et al, 1998). SULT2B1a also sulfates pregnenolone, while SULT2B1b sulfates cholesterol (Strott, 2002) . Furthermore, both failed to sulfate 4-nitrophenol or 17 -estradiol, classical substrates for the phenol and estrogen SULT subfamilies (Her et al, 1998). 1.4 Genes Expression and Regulation of Cytosolic SULTs Current knowledge of the regulation of SULT expression in humans is limited. Although most of the studies were carried out in animals, especially rodents, it is becoming more apparent that their regulation is somewhat different from that of humans. SULTs exhibit dramatic sexual dimorphism (Coughtrie et al, 1990; Wu et al, 2001). In addition, the complement and tissue distribution of the isoenzymes differs considerably between humans and animals. As a result, extrapolation of animal data to human must be done with careful consideration. Based on available reports, SULT isoforms show temporal and tissue-specific regulation. DNA sequences of SULT1A isoforms show the presence of multiple noncoding 5’-exons, which is thought to be involved in tissue-specific expression of these proteins (Weinshilboum et al, 1997; Dooley, 1998a). Rubin et al showed that SULT1A3 is essentially not expressed in the adult human liver but is very highly expressed in the gastrointestinal tract (Rubin et al, 1996). Furthermore, human SULT1A3 is known to be highly expressed in the fetal liver, with expression being “switched off” at around the time of birth (Capiello et al, 1991; Richard et al, 2001). SULT2A1 is not expressed at any developmental stage in the endometrium, but is highly expressed in the adrenal gland. In fact, it is more highly expressed in fetus than in adult (Barker et al, 1994; Rubin et al, 1999). SULT isoforms may also be regulated in a gender- and hormone-dependent manner. SULT1A1 is known as the “male dominant” enzyme of the SULT1A family; being more abundantly expressed in male relative to female rat liver. This age- and gender-related expression implies that pituitary factors other than growth hormones are probably involved in its regulation (Liu and Klassen, 1996). SULT1E1 protein expression varies widely between individuals. SULT1E1 was shown to be regulated in vivo by progesterone in addition to other factors, in the human endometrium using the menstrual cycle as a dependent variable. In vivo and in vitro studies with mice testis demonstrated that luteinising hormone was necessary and sufficient to maintain Leydig-cell SULT1E1 expression. The stimulatory effect of luteinising hormone on SULT1E1 expression in Leydig cells involves, at least partially, androgen action, further contributing to the complexity of its regulation (Falany and Falany, 1996; Rubin et al, 1999). More recently, SULT1E1 was implicated as an important factor in the regulation of breast-cancer cell (MCF-7) growth. Loss of SULT1E1 expression in the transformation of normal to tumourous cells increases the growth responsiveness of these cells to estrogen stimulation. Similarly, human SULT1E1 cDNA-transfected Ishikawa endometrial adenocarcinoma cells were 200-fold less sensitive to estradiol stimulation than control cells (Kotov et al, 1999; Falany et al, 2002). 1.5 Hepatic Vectorial Transport 1.5.1 An Overview The liver plays an important role in the detoxification of xenobiotics. Certain drugs are preferentially excreted into the venous outflow rather than into the bile. Endogenous compounds or xenobiotic uptake first occurs across the sinusoidal membrane into the hepatocytes by either simple passive diffusion or carrier-mediated transport systems as shown in Figure 1.5 (Takenaka et al, 1995; Milne et al, 1997). This is followed by intracellular transfer of the drug across the cytosol by transfer proteins that prevent refluxing of the chemical back through the sinusoidal membrane (Arias, 1976). Cytoplasmic proteins that have been shown to participate in intracellular transfer processes include glutathione S-transferases, fatty acid-binding proteins, and 3-alphahydroxysteroid dehydrogenase (LeBlanc, 1994). Movement of these compounds across the cytosol to the canalicular membrane possibly involves intracellular trafficking where these compounds migrate in vesicles along the microtubular network of the cell (Haussinger et al, 1993; Marks et al, 1995). These conjugated drugs as well as bile salts are then exported across the canalicular membrane into bile. Although bile is isoosmotic to plasma, bile salts are concentrated up to 1000-fold in bile, hence the need for active transport by the hepatocytes in their elimination (Meier and Stieger, 2002). Moreover, export of hydrophilic drug conjugates such as sulfated, glucuronidated and glutathioneconjugated compounds, require active transport for the elimination. Numerous transporters have since been cloned and characterized. These include isoforms of MRP (multidrug resistance-associated protein), isoforms of MDR (multidrug resistance) protein; both belonging to the superfamily of ATP-binding cassette (ABC) transporters, NTCP (sodium-taurocholate co-transporting polypeptide), BSEP (bile salt export pump), the OATPs (organic anion transporting polypeptides), OATs (organic anion transporters), and OCTs (organic cation transporters). Figure 1.5 Hepatic vectorial transport (Hooiveld et al, 2001) The sinusoidal (also termed as basolateral) uptake system involves the NTCP, OATPs, OCTs. Canalicular efflux system comprises the BSEP, MRPs and MDRs. 1.5.2 Hepatic Xenobiotic Uptake Transporters Generally, these transport systems can be broadly divided into two categories; the sodium-dependent and sodium-independent transport systems. Sodium-dependent transport is maintained by the sodium-potassium-ATPase of the basolateral plasma membrane (Kullak-Ublick, 2000). The sodium-dependent system is represented by the sodium-taurocholate cotransporting polypetides, NTCP (human) and ntcp (rat) [Figure 1.5]. NTCPs, located on the basolateral membrane, are exclusively expressed in the liver. The human NTCP is a 349- amino acid protein. NTCPs are unidirectional pumps that have been demonstrated to show preferential transport of sulfobromophthalein, bile salts, estrone-3-sulfate, ouabain, and other neutral steroids, as well as certain amphipathic organic cations. Although some unconjugated bile salts (e.g. deoxycholate, lithocholate) may enter the hepatocytes by nonionic diffusion, others (e.g. cholate) are predominantly taken up by the sodium-independent transport systems (HagenBuch and Meier, 1996; Haussinger et al, 2000). Sodium-independent uptake of bile salts is mediated by a variety of transporters, namely the organic anion transporting polypeptides (OATPs), the organic anion transporters (OATs) and the organic cation transporters (OCTs) (Kullak-Ublick et al, 2000). Members of the OATP family translocate a broad range of substrates. Presently, members of this family consist of oatp1, oatp2 and oatp4 in rat liver, and OATP-C (also called OATP6) and OATP8 in human liver, in addition to OATP-A, OATP-B, OATP-D and OATP-E (Noe et al, 1997; Reichel et al, 1999; Cattori et al, 2000; Kullak-Ublick et al, 2001). OATPs have been shown to translocate organic anions such as bromosulfophthalein and biotin, sulfated conjugates such as the sulphated steroid dehydroepiandrosterone (DHEA)-3-sulphate and also organic cations such as quinidine. In addition, it also transports neutral compounds such as ouabain (Kullak-Ublick et al, 1995; Kullak-Ublick et al, 1998; Kakyo et al, 1999; Konig et al, 2000). Studies with oatp1 displayed a broad spectrum of substrate specificities. In addition, it also showed overlapping substrate specificity to those transported by the NTCP, albeit at a much lower affinity. In contrast, it was shown that oatp1 has a very high affinity for estrone-3-sulphate and estradiol-17 -glucuronide, with Km values of about 11 µM and 4 µM respectively (Eckhardt et al, 1999). Recent findings suggest that uptake of anionic compounds by oatp1 is driven by the countertransport of intracellular glutathione (Lee et al, 1998; Lee et al, 2001). Hence, oatp1 is thought to represent a sinusoidal GSH efflux system. Homologues of the OATPs have been identified as OAT-K1 and OAT-K2; both cloned from the kidney. OAK-1 transports methotrexate and folate while OAK-2 transports methotrexate and folate, in addition to taurocholate and prostaglandin (Saito et al, 1996; Masuda et al, 1999). Translocation of organic cations (e.g. drugs, choline and monoamine neurotransmitters) is mediated by the organic cation transporters (OCTs). The transporters belong to a solute carrier family that comprises more that 18 different gene products. Presently, this OCT gene family comprises the OCT1/oct1, OCT2/oct2 and oct3, and the novel organic cation transporters; OCTN-1 and OCTN-2 as well as the organic anion transporters OAT1 to OAT3 (Takashi et al, 2000; Suzuki and Sugiyama, 2000; Sweet et al, 2001). 1.5.3 Hepatic Efflux Transporters Bile salts at the canalicular membrane are predominantly excreted by the bile salt export pumps; Bsep (rat) and BSEP (human); in an ATP-dependent manner (Stieger and Meier, 1998). BSEP is known to be a 160 kD-homologue of the Mdr gene product and was first characterized based on the transport of taurocholate (Gerd et al, 2000). BSEP has also been identified to be absent in patients with progressive familial intrahepatic cholestasis type 2, characterized by low biliary salt concentrations (Jansen et al, 1998). Also important for xenobiotic efflux are the two subclasses of ATP-binding cassette (ABC) superfamily of transporters, the multi-drug resistance-associated proteins (MRPs) and the P-glycoproteins (P-gps). ABC transporters are membrane proteins and their functions driven by ATP hydrolysis. Each ABC transporter typically consists of 12 or more membrane-spanning domains and two intracellular nucleotide-binding loops. The highly conserved nucleotide-binding domains contain the signature Walker A and B motifs, involved in the binding and hydrolysis of ATP (Klein et al, 1999). MRP1 is localized on the basolateral hepatocytes membrane. MRP1, the first isoform to be discovered, was shown to efflux glutathione S-conjugates such as leukotriene C4, steroid conjugates such as 17 -glucuronosyl-estradiol and glucuronidated or sulfated bile salt conjugates, divalent as well as monovalent bile salts. A homologue of MRP1 with quantitatively similar substrate specificity, called the MRP2 or canalicular multispecific organic anion transporter (cMOAT) is expressed on the canalicular membrane and transports bilirubin glucuronides. The subtrate spectrum for MRP2 is qualitatively similar to that of MRP1, but MRP2 does not transport monovalent bile salts (Buchler et al, 1996; Taniguchi et al, 1996; Jedlitchsky et al, 1996; Madon et al, 1997; Ito et al, 1998). MRP3 which is found on the basolateral hepatocytes membrane, have been shown to transport estradiol-17 -D-glucuronide and S-(2,4-dinitrophenyl-) glutathione, bile salts and certain anticancer drugs like methotrexate and etoposide (Stieger and Meier, 1998; Hirohashi et al, 1999; Kool et al, 1999). Unlike mrp1 and mrp2, mrp3 (rat) poorly transports glutathione S-conjugates (Hirohashi et al, 1999). The newer members, namely MRP4 and MRP5, transport anionic purine and nucleotide analogs like PMEA and 6-mercaptopurine (Kruh et al, 2001). A more recently discovered MRP6 was found on the basolateral and canalicular membrane of the hepatocytes, and is consitutively expressed. MRP6, which does not recognize MRP1/2 substrates, transports the glutathione S-conjugates leukotriene C(4) and S-(2, 4-dinitrophenyl)glutathione and the cyclopentapeptide BQ123 (an endothelin receptor antagonist) but not glucuronate conjugates such as 17 -estradiol 17-( -D-glucuronide). Studies suggest that MRP6 may not play a major role in xenobiotic detoxification. Rather, it was suggested to play a role on the transport of small peptides involved in cellular signaling and hormonal regulation of hepatocellular functions (Madon et al, 2000; Renes et al, 2000; Belinsky et al, 2002). It is observed that patients with Dubin-Johnson disease do not express MRP2 due to missense mutations or base deletions in the genes. This in turn leads to a compensatory increased expression of MRP3 in these patients. MRP2 is also significantly downregulated in cholestasis while MRP3 is upregulated. It is thought that this compensatory increase in MRP1 and MRP3 expression presumably prevents the hepatocytes from hepatotoxic injury by regulating the expression level of the individual bile salt transporters (Kartenbeck et al, 1996; Oswald et al, 1998). The P-gps are encoded by the human MDR1 or rodent mdr1 genes. These proteins are not only expressed in drug-resistant cells, but also at the apical domains of cells in normal tissues with excretory functions, such as the liver (at the canalicular hepatocytes membrane), small intestines (brush border membranes of enterocytes), kidney (brush border membrane of proximal tubule cells) and at the blood-brain barrier (capillary endothelial cells). MDR1/mdr1 has been shown to be important for the elimination of not only the typical MDR1 substrates, but also of relatively small aliphatic and aromatic cationic drugs such as tri-n-butylmethylammonium and azidoprocainamide (Smit et al, 1998). In addition, it has been proposed to also transport endogenous agents, such as steroid hormones (Karssen et al, 2001; Zampieri et al, 2002). In contrast, the liverspecific MDR3 (or Mdr2 in rats), functions as an ATP-dependent phosphatidylcholine translocase (Oude and Groen, 2000). Patients with progressive familial intrahepatic cholestasis type 3 carry mutations or deletions in the MDR3 gene and show absence of MDR3 expression. Serum bile salts and glutamyltranspeptidases are elevated in these patients (Jansen et al, 1999). 1.6 Effects of Glucocorticoids on Cytosolic SULTS and Xenobiotic Transporters Historically, these enzymes have been considered unresponsive to “classical” xenobiotic inducers. However, certain SULT isoforms have been shown to respond to phenobarbital, peroxisome proliferators and steroids, as well as natural dietary chemicals including polyphenols such as quercetin, components of red wine, and caffeine in green tea and coffee (Runge-Morris, 1998; Runge-Morris et al, 1998; Duanmu et al, 2000; Coughtrie et al, 2001). Wu et al previously reported that dexamethasone (DX) induced SULT1B1 expression in male but not female rats (Wu et al, 2001). Duanmu et al demonstrated that rat but not human hepatic SULT1A1 mRNA and protein expression were induced by DX in a concentration-dependent manner. However, rat and human SULT2A1 mRNA and protein expression increased in response to a similar DX treatment. PXR transcription factor was implicated in DX-induction of rat and human SULT2A gene expression. In addition, Duanmu et al also cloned and characterized the glucocorticoid (GC)-response element (GRE) of the rat SULT1A1, providing strong evidence of the involvement of GC in the regulation of the rat SULT1A1 gene (Duanmu et al, 2001; Duanmu et al, 2002). Treatment of cultured bovine tracheobronchial epithelial cells with hydrocortisone produced increased SULT1A1 enzyme activity and mRNA levels in a concentrationdependent manner. Similarly, the administration of pharmacological doses of DX (a potent GC) to rats increased SULT1A1 mRNA levels in both male and female rat liver (Beckmann et al, 1994). At the efflux level, less is known in relation to regulation by GCs. The antiestrogen tamoxifen, corticosteroid and DX were shown to induce mrp2 in rat and monkey hepatocytes (Kauffmann et al, 1998; Demeule et al, 1999). SULTs and transporters play a major role in the metabolism and transport of endogenous compounds and xenobiotics (including therapeutic drugs). As such, it is important to determine the factors and xenobiotics that can regulate expression levels and activities of SULTs and the transporters. This knowledge will have an important bearing on the biotransformation and transport of endogenous compounds and xenobiotics, which can potentially contribute to the refinement or development of therapeutic strategies in liver cholestasis and liver cancer. 2. Objective And Scope Of This Work Sulfation constitutes an important pathway for detoxification of xenobiotics. This process yields sulfated conjugates which are polar. Thus, transporters play an essential role in the elimination of these sulfated conjugates. This work aimed to explore the effects of glucocorticoids, DX and PN, on human SULT1A activity, PAPS synthesis (i.e. the ability of PAPSS to generate PAPS) and efflux capacity of sulfate conjugates using the human hepatocarcinoma cell line, Hep G2. The scope of this work is as outlined in Figure 2.1 below. X • PAPS generation assay • • SULT1A assay Reverse transcriptionpolymerase chain reaction (RT-PCR) Software analysis for regulatory elements of SULT1A3 gene ATP + APS PAPSS Nucleus X + PAPS ATP Hep G2 cell membrane • SULT1A1 SULT1A3 X-SO4 • • Efflux assay for sulfate conjugates RT-PCR of potential transporters X-SO4 Figure 2.1 Schematic outline of the scope of this work Following GC treatment on Hep G2, experiments were performed as listed in the dotted boxes on the right. “X” represents the substrates -nitrophenol for assay of SULT1A1 activity and dopamine for assay of SULT1A3 activity. 3. Materials and Methods 3.1 Materials All materials for cell culture were purchased from Gibco-BRL(USA). Lglutamine for cell culture was purchased from Sigma-Aldrich Pte. Ltd., USA. The 75 cm2 cell-culture flasks were obtained from Nunc, USA. Radiochemicals, Na235SO4 and PAP35S were purchased from NEN Life Sciences (USA). The specific activity of Na235SO4 used was 570.6 mCi/mmol; at 2 mCi/ml of radiochemical concentration. The specific activity of PAP35S used was 2.8 Ci/mmol; at 1.25 mCi/ml. All other chemicals used for cell-based assays were purchased from Sigma-Aldrich Pte. Ltd., USA. Access One-Step RT-PCR Kit was supplied by Promega, USA. North2South Direct HRP Labeling and Detection Kit was purchased from Pierce, USA. Hybond-N+ membrane for DNA transfer of SULT1A3 and the autoradiography films were purchased from Amersham Biosciences Ltd., USA. All other chemicals for RNA/DNA-based assays were purchased from Sigma-Aldrich Pte. Ltd., USA. 3.2 Methods 3.2.1 Cell Culture of Hep G2 Hep G2 was grown as a monolayer in 75cm2 culture flasks, in complete growth medium consisting of Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 1 mM sodium pyruvate and 0.1 mM MEM non-essential amino acids. The cells were cultured at 37 oC in a humidified atmosphere of 5% CO2. 3.2.2 Treatment Of Hep G2 With Glucocorticoids 0.2 x 107 cells were seeded per 75 cm2 flask (i.e. split ratio of 1:7). The cells were allowed to recover for 24 hours in the 37 oC humidified incubator supplemented with 5% CO2. On the following day, fresh culture medium each containing DX and prednisolone (PN) was added to the cultures at 10-5 M and 10-7 M (containing 0.02% DMSO final concentration). The cells were treated over 3 days. Treatment media were renewed after every 24 hours. Hep G2 cells grown in normal culture medium containing 0.02% DMSO were used as controls (Runge-Morris et al, 1999; Duanmu et al, 2001). For SULT1A assays using pre-conditioned Hep G2, the cells were cultured as described earlier using DMEM containing 10% FBS. Prior to experiment, the medium was replaced with DMEM containing 10% charcoal-stripped delipidated serum. The cells were grown at 37 oC in a 5% CO2 humidified incubator for 16 hrs before addition of glucocorticoids (Pacussi et al, 2001). The cells were harvested by trypsinization with trypsin-EDTA, resuspended in phosphate-buffered saline (PBS) pH 7.4, and centrifuged for 5 minutes at 2000 g. The pellet was then washed in PBS and re-centrifuged as before. The final pellet was resuspended in PBS and kept frozen at -80oC. Freezing followed by thawing of the samples twice resulted in the release of the cell contents and these lysates were then used for enzymatic assays. Protein concentration was determined using BioRad Protein dye, based on the method of Bradford with bovine serum albumin as the standard (Bradford, 1976). For studies on efflux of sulfated conjugates, the cells were trypsinized and centrifuged in PBS as described above. The pellet was then washed in Hank’s Balanced Salt Solution (HBSS) pH 7.4. The final pellet was resuspended in HBSS and kept on ice throughout the experiments. The buffer compositions for PBS and HBSS are as listed in Table 3.1 below. Buffer PBS Composition 0.14 g/L KH2PO4, 9.0 g/L NaCl, 0.8 g/L and Na2HPO4, pH 7.4 HBSS 5.8 mM K+, 143 mM Na+, 1.3 mM Ca++, 0.8 mM Mg++, 146 mM Cl-, 0.8 mM Pi, 4.2 mM HCO3-, 5.6 mM glucose and 10mM Hepes, pH 7.4 Table 3.1 Buffer compositions of PBS and HBSS 3.2.3 Cell viability Following cell harvest, a cell count was done to ensure that cell growth was unaltered following treatment. Cell viability was assessed using trypan-blue exclusion method. After trypsinization of the cells in 75cm2 flasks as described in earlier in Section 3.2.2, the cells were resuspended to a final volume of 10 ml with PBS of which 100 µl was removed to a sterile microfuge tube for cell counting and cell viability assessment. The remaining cell suspension was then washed and re-centrifuged for enzymatic assays as described earlier in Section 3.2.2. 3.2.4 Assay of SULT1A1 and SULT1A3 Activities In Hep G2 -Nitrophenol was used as the substrate to assay for SULT1A1 activity and dopamine for SULT1A3 activity. Each 120 µl reaction incubate contained one of the substrates: 10 µM dopamine or 4 µM -nitrophenol, with 2.25 µM PAP35S and cell lysate containing 120 µg protein, buffered at pH 6.5 using 50 mM potassium phosphate (KH2PO4). The reaction was incubated for 3 min at 37oC, as was previously described (Frame et al, 2000). For assays with dopamine as the substrate, the cell lysate was preincubated with 1 mM trans-2-phenylcyclopropylamine (a monoamine oxidase inhibitor) for 15 min at 37oC, as described previously (Wong and Wong, 1996). Each reaction was terminated by adding 18 µl each of 0.3 M zinc sulfate, ZnSO4 and 0.3 M barium hydroxide, Ba(OH)2. The mixture was mixed thoroughly and then clarified by centrifuging at 20,000 g for 4 min. The clarified supernatant was filtered through a 0.45 µm membrane followed by HPLC-radiometric detection for the sulfated conjugates (Wong KP, 1976). 3.2.5 Efflux Assays Of Sulfated Conjugates Of Dopamine and -Nitrophenol A final reaction volume of 250 ul containing 250 µg protein, 100 µM Na235SO4, 150 µM dopamine or 2 µM -nitrophenol and buffered at pH 7.4 with HBSS, was incubated at 37 oC for 30 min. In assays with dopamine as the substrate, monoamine oxidase in the cells was inhibited with a 15-min pre-incubation of the cell suspension with 1 mM trans2-phenylcyclopropylamine at 37 oC. The reaction was terminated by centrifuging 180 µl of the sample through a layer of oil mixture at 20,000 g at room temperature for 4 min. The oil mixture consists of bath fluid (density of 1.05) and mineral oil (density of 0.84) in a ratio of 9.05 to 0.95 (Studenberg and Brouwer, 1993). Following centrifugation, 90 µl of the upper aqueous layer was then clarified by adding 9 µl each of 0.3 M ZnSO4 and 0.3 M Ba(OH)2. After thorough mixing, the sample was centrifuged again at 20,000 g at room temperature for 4 min. The precipitate was discarded and supernatant was filtered through a 0.45 µm membrane filter for analysis by HPLC-radiometric detection for the sulfated conjugates. 3.2.6 Reverse-phase High-performance Liquid Chromatography (RP-HPLC) Detection And Separation Of The Sulfated Conjugates Of Dopamine And Nitrophenol From Na235SO4 Solvent compositions for the separation of dopamine- and -nitrophenyl-sulfate conjugates are as listed in Table 3.2 below. The solvent was delivered at 1 ml/min flow rate through a Hypersil ODS column (4.6 x 200 mm, 5 µm pore size). Solvent delivery and sample injections were performed using HP1100 series of quarternary pump and autosampler (Hewlett Packard, USA), and the radiometric detector used was Flo-one Beta (Packard, USA) (Wong and Wong; 1994; Kuhn et al, 2001). Substrate -nitrophenol Dopamine Table 3.2 Buffer components % Buffer % Absolute Methanol 75 µM NaH2PO4 containing 1 µM EDTA, 0.35 mM 1-octanesulfonic acid pH 4, with 4% acetonitrile 89 11 As above 98 2 Solvent composition for the separation of dopamine- and -nitrophenylsulfate from Na235SO4 3.2.7 Assay Of PAPSS Activity (PAPS Generation Assay) Cell lysate containing 300 µg protein was co-incubated with 120 µM Na235SO4, 9.3 mM MgCl2, 9.3 mM ATP and 2 mM DTT in a total volume of 120 µl buffered at pH 7.5 with 50 mM KH2PO4, for 5 min at 37 oC. The reaction was terminated by boiling for 1 min. The precipitate was centrifuged at 20,000 g for 4 min at room temperature and the filtered supernatant was used for HPLC-radiometric detection of PAP35S. Detection of PAP35S was achieved using a NovaPak C18 column (3.9 x 150 mm, 4 µm pore size). The mobile phase consisted of 4 mM tetrabutylammonium perchlorate and 30 mM KH2PO4 buffer with 20% methanol, pH 7.0. The flow rate was 1 ml/min (Wong and Wong, 1996). 3.2.8 Statistical Analysis Results are presented as mean + standard deviation (SD) of triplicates from at least two individual experiments. Comparison of the different treatment groups was carried out using ANOVA followed by a Tukey’s post-hoc test. A p-value of 107859 /organism="Homo sapiens" /mol_type="genomic DNA" /db_xref="taxon:9606" /clone="RP11-347C12" /note="Accession AC106782 sequenced by Joint Genome Initiative, U.S. Dept. of Energy" source 60) at this position. Consensus index (ci) for the matrix represents the degree of conservation of each position within the matrix. The maximum ci-value of 100 is reached by a position with total conservation of one nucleotide, whereas the minimum value of 0 only occurs at a position with equal distribution of all four nucleotides and caps (MatInspector, Genomatix, Germany). AP1 2469 Figure 4.18 SP1 1629 1624 AP2 GRE CAAT 1249 1204 184 177 +1 Putative regulatory factors and elements of the human SULT1A3 gene ‘+1’ denotes the transcription start site of SULT1A3 gene which is defined at 4801 bp position on NT_024812 Region: 1605606 – 1713464. 5. Discussion 5.1 Effects Of Glucocorticoids On Sulfation In Hep G2 Cells The human hepatoma cell line, Hep G2 was used in this study. This cell line had been shown to have sulfation capacity (Shwed et al, 1992; Walle et al, 1994) and recent work had shown that it is also capable of exporting a series of sulfated conjugates (Ng et al, 2003). Results from this work are in agreement with these observations. Hep G2 cells constitutively expressed SULT1A1 and SULT1A3 activities. Using -nitrophenol and dopamine as the representative substrates for SULT1A1 and 1A3 respectively, Hep G2 cells were able to form the corresponding sulfated metabolites and net efflux of these metabolites was observed. This study was carried out to examine the effects of the glucocorticoids, DX and PN on the conjugation process, as well as on the efflux of sulfated conjugates. Results show that DX affects the sulfation process via the SULTs, while PAPS generation was unaffected i.e. PAPSS activity was not affected. However, only SULT1A3 but not SULT1A1 responded to DX. On the other hand, PN under similar experimental conditions did not alter both PAPSS and SULT1A activities. These results imply that not all glucocorticoids affect SULT1A isoforms in the same manner. DX and PN are the chemically modified versions of the naturally occurring hormone, cortisol (as known as hydrocortisone). As illustrated in the following figures (Figures 5.1), PN differs from DX at position C-9 where a fluorine atom is absent, and position C-16, where a methyl substitution is absent. These differences in the chemical structure make DX more potent that PN. In terms of anti-inflammatory potency, DX is about six times more potent that PN. Thus, using the same concentrations of DX and PN in the treatment of the hepatocytes, it is possible to obtain a GC-inducible effect on SULT1A3 in Hep G2 cells with DX but not with PN. A Figure 5.1 B (A) Prednisolone and (B) Dexamethasone Presence of a fluorine atom in position C-9 of dexamethasone increases its anti-inflammatory potency and the methyl group at position C-16 decreases its mineralocorticoid activity. 5.2 DX Differentially Induces SULT1A3 But Not SULT1A1 Activity Interestingly, SULT1A1 did not respond to DX at both concentrations. Indeed the lack of SULT1A1 response agrees well with those of Duanmu et al who had recently reported that DX-inducible rat SULT1A1 gene expression is not conserved in human (Duanmu et al, 2002). Studies of DX-inducible expression of CYP3A and SULT1A1 genes are routinely carried out at 10-7 M DX, which represents the physiological concentration, and at the supramicromolar, 10-5 M DX (Seree et a, 1998; Sumida et al, 1999, Duanmu et al, 2001). Treatment with the lower, physiological concentration of 10-7 M DX has consistently resulted in a greater induction of SULT1A3 activity (Figure 4.5B), which correlated with its increased mRNA level (Figure 4.10). Treatment with the 100-fold higher 10-5 M DX only produced little or no effect on SULT1A3 activity. This response was also observed for Hep G2 cells which were pre-conditioned in culture medium containing charcoalstripped delipidated serum (Figure 4.7B). In rat hepatocytes, similar observations of higher induction of aryl sulfotransferase IV (rat SULT1A1) and tyrosine amino transferase at 10-7 M DX compared to 10-5 M had been reported (Runge-Morris et al, 1998). To date, it is unclear why this occurs. Over the past decade, numerous steroid receptors (e.g. glucocorticoid receptor [GR], pregnane-X receptor [PXR], farnesoid-X receptor [FXR] and retinoic-X receptor [RXR]) and nuclear orphan receptors (such as chicken ovalbumin upstream promoter transcription factor [COUP-TF], hepatic nuclear factor 4 [HNF4]) have been shown to regulate cytochrome P450 enzymes though the exact mechanisms are not fully elucidated (reviewed in Quattrochi and Guzelian, 2001). Recently, Pacussi et al reported that submicromolar concentrations of DX resulted in a direct GR-mediated up-regulation of the steroid receptors, PXR and constitutive androstane receptor (CAR), which consequently up-regulated CYP3A4 gene transcription in a DX-independent manner. On the other hand, DX at supramicromolar concentrations (>10-5 M) serve as a ligand and an activator of PXR; resulting in direct activation of PXR (Pacussi et al, 2001). Similarly, current data from rat hepatocytes also indicate that DX-inducible SULT2A1 expression may be regulated via both PXR and non-PXR mediated mechanisms (Duanmu et al, 2002). Present results from this work strongly indicate the involvement of the GRmediated pathway in the regulation of human SULT1A3 gene. MatInspector sequence analysis of the promoter region revealed the presence of a GRE approximately 1.2 kb upstream of the transcription start site of the SULT1A3 gene. Further experimental evidence employing reporter gene assays will serve to confirm current observation. Using deletion constructs of the rat SULT1A1 5’-flanking region, Duanmu and coworkers showed that DX-induction of promoter constructs containing up to 1893 bases upstream of the transcription start site produced a 1.5-fold higher response with 10-7 M relative to induction with 10-5 M. However, constructs containing only 119 bases upstream of transcription start site resulted in similar levels of induction at both 10-5 M and 10-7 M (Duanmu et al, 2001). The magnitude of the shorter construct was much reduced; suggesting that there may be at least one cis-acting element located in this region responsible for this observation. For future experiments in this study, it would be logical to make a series of deletion constructs of the promoter region of SULT1A3 spanning the region where the GRE was predicted. Promoter activity analysis of these constructs would reveal the GC-responsive region(s) of the promoter, as well as the involvement of the putative GRE in SULT1A3 gene expression. 5.3 Effects of DX On Efflux Of Sulfated Conjugates In Hep G2 Cells Efflux assays show that -nitrophenyl-sulfate efflux was decreased by nearly 50% while efflux of dopamine-sulfate was increased by 150% at the physiological concentration of 10-7 M DX. This opposing response clearly demonstrates that the transporter(s) for -nitrophenyl-sulfate is likely to be different from that of dopaminesulfate. Moreover, these results also show that the transporters for these sulfatedconjugates are responsive to DX. Although the transporters for these sulfated conjugates have not been identified, it is likely that MRP-like transporters are responsible for the cellular efflux of p-nitrophenyl sulfate and dopamine sulfate while members of the OATP family are capable of facilitating the uptake of these sulfated compounds (Ng et al, 2003). Effects of DX on members of the MRP and OATP transporter families are not well studied. MRP2, MRP3, MRP5 and OATP-C have all been shown to be induced by various xenobiotics including phenobarbital and rifampicin (Zhu and Center, 1994; Kast et al, 2002; Tirona et al, 2003). The effects of rifampicin are mediated by the pregnane X receptor (PXR) (Kast et al, 2002; Tirona et al, 2003). Present results show that DX generally induces the MRP isoforms except for MRP2 and MRP3. MRP3 mRNA expression was decreased. MRP2 mRNA expression was decreased only at 10-7 M but increased at supramicrolar concentration of 10-5 M. This biphasic response is similar to that reported for the rat GSTA2 (Falkner et al, 2001). MRP2 has also been shown to be induced in human hepatocytes and HepG2 cells following treatment with rifampicin, a PXR ligand (Schrenk et al, 2001). Present results from this work perhaps also suggest the involvement of PXR-mediated regulation since supramicromolar DX concentrations also activate PXR (Pacussi et al, 2001). MRP1 mRNA expression was also DX-inducible (Table 4.2). As shown in Figure 5.2, the 5’-untranslated region of human MRP1 was found to contain putative binding sites for various response elements; including a putative GC response element [GRE] (Zhu and Center, 1994). However, no report on the mechanism of regulation of MRP1 by DX (or other GC’s) is available as yet. Nevertheless, it has been shown with cultured hepatocytes, that MRP1 is at least partially regulated by reduction-oxidation status although the exact underlying mechanisms are still unresolved (Yamane et al, 1998; Roelofsen et al, 1999). -1000 CRE Figure 5.2 -600 ERE -400 AP1 ERE -200 GRE +1 AP2 AP2 SP1 SP1 Potential response elements in the 5’-untranslated region of human MRP1 (http://www/med.rug.nl/mgl/promoter.htm) MRP3 mRNA level was decreased in response to DX. Previously, MRP3 was shown to be responsive to xenobiotics that are constitutive androstane receptor (CAR) activators such as phenobarbital, and the microsomal enzyme inducer, diallyl sulfide (Ogawa et al, 2000; Cherrington et al, 2002). Present results show that at high DX concentration (which can activate PXR), MRP3 mRNA expression is decreased. 5.4 Effects of DX On Detoxification Via Sulfation Pathway Essentially, present results indicate that DX affects sulfation in Hep G2 cells by inducing SULT1A3 gene expression and protein activity, but does not affect the ability of PAPSS to generate PAPS. The decreased efflux of -nitrophenyl-sulfate can be due in part to increased OATP expression (Table 4.2) or reduced efflux of the conjugate, or both; but is unlikely be due to a change at the sulfation level since SULT1A1 activity was not responsive to DX. However, increased efflux of dopamine-sulfate at 10-7 M DX is likely a result of increased SULT1A3 activity and up-regulation of its efflux. 6. Conclusion DX induces SULT1A3 but not SULT1A1 and PAPSS activity in Hep G2 cells. PN did not produce any significant alterations to SULT1A activity. RT-PCR revealed that mRNA expression of the OATP and MRP isoforms were generally increased in the presence of DX, except for MRP3 which was repressed, while a biphasic response was observed for MRP2. Efflux of -nitrophenyl-sulfate was down-regulated by DX by nearly 50%; probably due to increased uptake, possibly by OATP proteins and/or reduced export. Dopamine-sulfate efflux was up-regulated by 150% at 10-7 M DX; probably a result of increased efflux in addition to the increased SULT1A3 activity. Current results show that glucocorticoid, dexamethasone influences the enzymatic activity and gene expression of human SULT1A1, as well as the gene expression of the major hepatic xenobiotic transporters. These findings in addition to more in-depth studies, will potentially contribute to the undertstanding of their gene regulation and the development of better therapeutic strategies for liver cholestasis and cancer. 7. Future Work Sequence analysis of the putative promoter region indicates that the region up to ~3 kb proximal to the transcription start site may contain several important responsive elements. Reporter gene assays using deletion constructs spanning this ~3-kb region will help identify crucial regions involved in the human SULT1A3 gene. The above data showed that DX could affect the mRNA levels as well as the enzymatic activities of SULT1A3. A computer-based search of the 5’ flanking region of SULT1A3 gene showed the presence of a consensus binding site for the GC receptor. Such a site was not present for SULT1A1. To date, there had been three other studies on the effects of xenobiotics on SULT activities. Two of these showed the induction of SULT activity towards 4-nitrophenol and 17 -ethinylestradiol by rifampicin while the third showed induction of SULT2A1 by DX (Kern et al, 1997; Li et al, 1999; Duanmu et al, 2002). More work will have to be done to define the regulatory elements involved and to determine the role of transcriptional factors and nuclear receptors. Work will also have to be carried out to address the effect on other SULT genes, as well as the effect on candidate transporters, namely members of the OATP and MRP families. The xenobiotics used in this study is representative of only one class of compounds (i.e., GC’s) known to modulate the expression of enzymes involved in drug metabolism. Other compounds including barbiturates (e.g. phenobarbital), pregnanes (e.g. pregnenolone 16 -carbonite), steroidal compounds (e.g. RU486, spironolactone, dihydrotestosterone, 17 -ethinyloestradiol and 17 -estradiol) and macrolide antibiotics (e.g. rifampicin) have all been shown to modulate cytochrome P450 genes and some Phase II drug metabolism genes. 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Abbreviations 5’-UTR 5’-untranslated region APS adenosine-5’-phosphosulfate CAR constitutive androstane receptor COUP-TF chicken ovalbumin upstream promoter transcription factor DHEA-ST dehydroepiandrosterone sulfotransferase DMEM Dulbecco’s Minimum Essential Medium DX dexamethasone FBS fetal bovine serum FXR farnesoid-X receptor GC glucocorticoid GR glucocorticoid receptor GRE glucocorticoid response element GSTA2 glutathione S-transferase A2 HBSS Hank’s Buffered Salt Solution HNF-4 hepatic nuclear factor-4 HRP horseradish-peroxidase MRP multidrug resistance-associated protein OAT organic anion transporter OATP organic anion transporting polypeptide PAPS 3’-phosphoadenosine 5’-phosphosulfate PAPSS 3’-phosphoadenosine 5’-phosphosulfate synthetase PBS Phosphate Buffered Saline PN prednisolone PXR pregnane-X receptor RT-PCR reverse-transcription polymerase chain reaction RXR retinoic-X receptor SDS sodium dodecyl sulfate SULT sulfotransferase [...]... 5’phosphate group in the PAPS molecule The hydroxyl side-chain of the conserved serine interacts with an O-atom in the 3’-phosphate X-ray crystallography of hEST also showed that the side-chain of the conserved Ser137 interacted with the side-chain of the conserved K47 As a result of the interaction, the side-chain nitrogen of the conserved lysine is repelled from the bridging oxygen of the PAPS molecule... up of 12 exons, but exon 1 (the first splice junction) contains an additional codon in PAPSS2 All exon-intron splice sites for the two genes are virtually identical Introns of PAPSS1 vary from 1.6 kb to 21.9 kb whereas the introns of PAPSS2 are generally shorter than those of PAPSS1 Table 1.2 summarizes the characteristics of human PAPSS1 and PAPSS2 genes The 5’-flanking region of PAPSS1 did not contain... al, 2001) In addition, the complement and tissue distribution of the isoenzymes differs considerably between humans and animals As a result, extrapolation of animal data to human must be done with careful consideration Based on available reports, SULT isoforms show temporal and tissue-specific regulation DNA sequences of SULT1A isoforms show the presence of multiple noncoding 5’-exons, which is thought... glucocorticoid (GC)-response element (GRE) of the rat SULT1A1, providing strong evidence of the involvement of GC in the regulation of the rat SULT1A1 gene (Duanmu et al, 2001; Duanmu et al, 2002) Treatment of cultured bovine tracheobronchial epithelial cells with hydrocortisone produced increased SULT1A1 enzyme activity and mRNA levels in a concentrationdependent manner Similarly, the administration of pharmacological... activity and the binding of 35S-PAPS as a photoaffinity ligand for the enzyme (Komatsu et al, 1994) Furthermore, similar studies with SULTs in plants had led to the conclusion that the invariant lysine within Region I might be important for the stabilization of an intermediate formed during the sulfonation reaction (Marsolais and Varin, 1995) Figure 1.2 (next page) The highly conserved Region I and IV... the termini of the protein sequence; one being near the amino terminus (Region I) and the other near the carboxy terminus (Region IV) Through the cloning of SULT cDNAs, the consensus sequence that has been identified in Region I is YPKSGTxW and in Region IV is RKGxxGDWKNxFT, where “x” represents any amino acid The motif of Region IV is similar to the glycine-rich phosphate-binding loop (the “P-loop”),... signifies the role of PAPS synthetase in the generation of PAPS for sulfation 1.2.3 Cytosolic Sulfotransferases The sulfotransferases (SULTs) constitute a diverse range of enzymes that make up an emerging superfamily Historically, the reactions catalyzed by these low-capacity enzymes have been termed “sulfation”, although chemically, they are more accurately described as sulfonation Sulfonation/sulfation... hence the need for active transport by the hepatocytes in their elimination (Meier and Stieger, 2002) Moreover, export of hydrophilic drug conjugates such as sulfated, glucuronidated and glutathioneconjugated compounds, require active transport for the elimination Numerous transporters have since been cloned and characterized These include isoforms of MRP (multidrug resistance-associated protein), isoforms... Fatty acid conjugation -OH Condensation Various Table 1.1 Phase II conjugation reactions (Kauffman, 1990) 1.2 Sulfation 1.2.1 Sulfation: An Overview Sulfation plays a role in homeostasis and regulation of many important endogenous chemicals such as catecholamines, steroids as well as other macromolecules (Coughtrie et al, 1998) In addition, it serves as one of the detoxification pathways for the various... occasionally it results in the activation of the xenobiotic to a reactive electrophile (Buhl A et al, 1990; Falany, 1991; Glatt, 1997) The energy-requiring, sulfation process is catalysed by the substrate-specific sulfotransferases, using 3’-phosphoadenosine 5’-phosphosulfate (PAPS) and ATP as cosubstrates for the sulfation reaction Sulfation reactions utilize PAPS as the sulfate donor PAPS is made in the ... On SULT1A1 And SULT1A3 Activities 4.5 RT-PCR Detection: Effect Of DX And PN On SULT1A3 mRNA Expression 4.6 PAPS Generation Assay: Effect of DX and PN On SULT1A1 And SULT1A3 Activities 4.7 Efflux. . .EFFECTS OF XENOBIOTICS ON SULFOTRANSFERASE 1A (SULT1A) ACTIVITIES AND THE EFFLUX OF SULFATED CONJUGATES SHERRY NGO YAN YAN (BSc Biochemistry, Massey University, New Zealand) A THESIS SUBMITTED... essential role in the elimination of these sulfated conjugates This work aimed to explore the effects of glucocorticoids, DX and PN, on human SULT1A activity, PAPS synthesis (i.e the ability of PAPSS