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

1.3.1 An Overview 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 Xeno

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

1.3.1 An Overview

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

2 Objective And Scope Of This Work

3 Materials and Methods

3.1 Materials

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

3.2.8 Statistical Analysis 3.2.9 RNA Isolation And Reverse-transcription

(RT)-PCR Of SULT1A Isoforms And

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Xenobiotic Transporters

3.2.10 RT-PCR of SULT1A3 Followed By Chemiluminescence Detection

4 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

List of Figures

Figure 1.1 Human PAPSS1 and PAPSS2

Figure 1.2 (next page) The highly conserved Region I and IV amino

acid SULT signature sequences Figure 1.3 Proposed reaction mechanism of sulfuryl transfer

catalyzed by SULTs Figure 1.4 The human SULT enzyme family

Figure 1.5 Hepatic vectorial transport

Figure 2.1 Schematic outline of the scope of this work

Figure 4.1 Separation of -nitrophenyl-35sulfate from sodium-sulfate

(Na235SO4) Figure 4.2 Separation of dopamine-35sulfate from Na235SO4

Figure 4.3 Separation of PAP35S from Na235SO4

Figure 4.4 Time-dependent (A) -nitrophenyl-ST and (B)

dopamine-ST activities in Hep G2 Figure 4.5 (A) SULT1A1 and (B) SULT1A3 activities in Hep G2

following three days of DX treatment Figure 4.6 (A) SULT1A1 and (B) SULT1A3 activities in Hep G2

following three days of PN treatment Figure 4.7 (A) SULT1A1 and (B) SULT1A3 activities in pre-

conditioned Hep G2 cells prior to three days of DX treatment

Figure 4.8 (A) SULT1A1 and (B) SULT1A3 activities in

pre-conditioned Hep G2 cells prior to three days of PN treatment

Figure 4.9 RT-PCR of SULT1A3 and -actin in Hep G2 cells

following three days of GC treatment

Figure 4.10 Panel A: SULT1A3 mRNA levels in Hep G2 following

three days of GC treatment Figure 4.11 PAPS generation by PAPSS in Hep G2 following three

days of DX and PN treatment Figure 4.12 Efflux of (A) -nitrophenyl-35sulfate and (B) dopamine-

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transporters from total RNA extract of DX-treated Hep G2 cells

Figure 4.14 RT-PCR of various isoforms of OATP transporters

from total RNA extract of DX-treated Hep G2 cells Figure 4.15 SULT1A3 cDNA sequence (GenBank Accession Number:

U20499) Figure 4.16 Annotations of the human SULT1A3 genomic sequence

(GenBank Accession Number: NT_042812) Figure 4.17 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 Figure 4.18 Putative regulatory factors and elements of the human

SULT1A3 gene Figure 5.1 (A) Prednisolone and (B) Dexamethasone

Figure 5.2 Potential response elements in the 5’-untranslated region

List of Tables

Table 1.1 Phase II conjugation reactions

Table 1.2 Characteristics of human PAPSS1 and PAPSS2

Table 1.3 Names of the corresponding SULTs that are listed in

Figure 1.2, based on the new nomenclature and their GenBank Accession Numbers

Table 3.1 Buffer compositions of PBS and HBSS

Table 3.2 Solvent composition for the separation of dopamine-

and -nitrophenyl-sulfate from Na235SO4Table 3.3 Primer sequences of various isoforms of SULT 1A,

MRP, OATP and the control, -actin for RT-PCR Table 4.1 Typical cell concentration and viability of Hep G2

following three days of GC treatment Table 4.2 Intensities of SULT1A3 blot following

chemiluminescence detection of RT-PCR products Table 4.3 mRNA levels of the various transporters in DX-treated

Table 4.4 Consenus sequences of SP1, AP1, AP2, CAAT and

GRE used by MatInspector software

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 bio-

inactivation 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

Methyltransferase Acetyltransferase

Glutathione-S-transferase

-OH, -COOH, -NH2, -SH

-OH, -COOH, -SH -NH2, -SO2NH2, -OH -OH, -NH2

-NH2, -SO2NH2, -OH -COOH

Epoxide, Organic halide -OH

(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

ATP-sulfurylase 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)

Slice junctions conform to ‘GT-AG’ rule

Low expression in liver, skeletal muscle and

Several putative Sp1 binding sites at flanking region

5’-Slice junctions conform to ‘GT-AG’ rule Highly expressed in liver 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

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

Old Name New Name Accession No rPST

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

Table 1.3 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 side-chain 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, 5-

hydroxytryptamine, 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-acetylaminofluorene-specific 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

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 non-coding 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

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 hydroxysteroid 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

3-alpha-(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 glutathione-conjugated 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,

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

liver-specific 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 culturedbovine tracheobronchial epithelial cells with hydrocortisone producedincreased SULT1A1 enzyme activity and mRNA levels in a concentration-dependent manner Similarly,the administration of pharmacological doses of DX (a potent GC) to rats increased SULT1A1 mRNA levels in both male and femalerat 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

PAPSS ATP + APS

X + PAPS

ATP

X

SULT1A1 SULT1A3

regulatory elements of SULT1A3 gene

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

dopamine for assay of SULT1A3 activity

3 Materials and Methods

3.1 Materials

All materials for cell culture were purchased from Gibco-BRL(USA)

L-glutamine for cell culture was purchased from Sigma-Aldrich Pte Ltd., USA The 75 cm2cell-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