Department of Molecular & Cellular Pathology, University of Dundee, Ninewells Hospital & Medical School, Dundee, Scotland, UK

Correspondence to: MWH Coughtrie, Department of Molecular & Cellular Pathology, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland, UK. Tel: +44 (0)1382 632510; Fax: +44 (0)1382 640320; E-mail: m.w.h.coughtrie@dundee.ac.uk

Members of the cytosolic sulfotransferase (SULT) superfamily catalyse the sulfation of a multitude of xenobiotics, hormones and neurotransmitters. Humans have at least 10 functional SULT genes, and a number of recent advances reviewed here have furthered our understanding of SULT function. Analysis of expression patterns has shown that sulfotransferases are highly expressed in the fetus, and SULTs may in fact be a major detoxification enzyme system in the developing human. The X-ray crystal structures of three SULTs have been solved and combined with mutagenesis experiments and molecular modelling, they have provided the first clues as to the factors that govern the unique substrate specificities of some of these enzymes. In the future these and other studies will facilitate prediction of the fate of chemicals metabolised by sulfation. Variation in sulfation capacity may be important in determining an individual's response to xenobiotics, and there has been an explosion in information on sulfotransferase polymorphisms and their functional consequences, including the influence of SULT1A1 genotype on susceptibility to colorectal and breast cancer. Finally, the first gene knockout experiments with SULTs have recently been described, with the generation of estrogen sulfotransferase deficient mice in which reproductive capacity is compromised. Our improved understanding of these enzymes will have significant benefits in such diverse areas as drug design and development, cancer susceptibility, reproduction and development.

The Pharmacogenomics Journal (2002) 2, 297-308. doi:10.1038/sj.tpj.6500117

sulfotransferase; sulfation; pharmacogenetics; structural biology; development

SULT, Sulfotransferase; PAPS, 3'-phosphoadenosine 5'-phosphosulfate

Sulfation of a vast array of natural and synthetic chemicals as well as many biomolecules occurs widely in nature, from bacteria to humans. This article concerns the formation of sulfate conjugates of xenobiotic and endogenous (endobiotic) small molecules by members of the cytosolic sulfotransferase (SULT) enzyme family. The process was first discovered in the late 1870s by Eugen Baumann, who isolated and characterised the sulfate conjugate of phenol from urine of a patient treated with carbolic acid as an antiseptic. It would be another 80 years, however, before the mechanistic basis of sulfate conjugation could be identified, with the discovery of so-called 'active sulfate' by Lipmann's group.¹ This compound, that we now call PAPS (3'-phosphoadenosine 5'-phosphosulfate), is the sulfuryl donor for the vast majority of sulfation reactions² (Box 1).

Recent research in this field that will be highlighted here, has uncovered some important aspects of the function of sulfation. The relative importance of sulfation in humans has perhaps been underestimated in the past, and one of the main reasons for this is that there are major inter-species differences in the function of sulfation¾in particular relating to its role in modulating the function of hormones and neurotransmitters. Importantly, catecholamines, estrogens, iodothyronines and dehydroepiandrosterone (DHEA) exist in the human circulation predominantly or significantly as the sulfated form, whereas this is not the case for rodents or many other 'experimental' animals.³ This is reflected in the different SULT isoenzyme profiles that exist in various species. For example, to date human is the only species in which a catecholamine-specific SULT isoform (SULT1A3) has been identified, consistent with the high circulating levels of catecholamine sulfates found in humans and other primates.^3,4 Rodents have multiple isoforms of the hydroxysteroid SULT subfamily 2A,^5,6,7 whereas so far only a single SULT2A enzyme has been identified and characterised in humans.⁸ Also, it is now clear that SULTs are abundantly expressed in the human fetus^{9,10,11,12,13} ¾other 'drug metabolising enzymes' such as the cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UGTs) etc are, in general not expressed at significant levels until after birth^14,15 ¾thus SULTs may represent a front line of chemical defence in the developing human.

This article aims to provide the reader with an up-to-date overview of sulfation in humans, and to highlight some important recent research that has impacted significantly on our knowledge and understanding of this important metabolic system.

THE SULFOTRANSFERASE SUPERFAMILY¾ORGANISATION AND FUNCTION

As with many drug metabolising enzymes, the cytosolic SULTs are derived from a large superfamily of genes. Full-length cDNAs encoding more than 50 mammalian and avian cytosolic SULTs have been cloned and sequenced, and many of the expressed proteins characterised. The majority of these enzymes sulfate small molecule xenobiotics and/or endogenous hormones and neurotransmitters, and based on amino acid sequence analysis can be subdivided into six families (Box 2). In addition plants possess a number of SULTs related to the mammalian cytosolic enzymes, that are involved in flavonol and brassinosteroid metabolism.¹⁶ Other model organisms whose genomes have been (or are being) sequenced possess various SULTs¾the zebrafish (Danio rerio) and amphibian (Xenopus laevis, Silurana tropicalis) sequence databases contain numerous cytosolic SULT sequences with orthologs in mammalian species, although the Drosophila melanogaster, Caenorhabditis elegans and Saccharomyces cerevisiae databanks are almost devoid of such sequences. A new nomenclature system for the cytosolic SULTs has recently been developed* which will (hopefully) help to clarify the relationships between the various SULTs for novices and aficionados alike. The nomenclature used here follows this new system.

The SULT1 and SULT2 families are the largest, and probably the most important for xenobiotic and endobiotic metabolism. At present the human SULT enzyme family, which is the best characterised, comprises of 11 isoforms representing the SULT1, SULT2 and SULT4 families. These enzymes are the products of 10 genes (SULTs 2B1a and 2B1b are generated by alternate splicing of the first exon of SULT2B1) that share many common structural features (reviewed in¹⁷). The properties of the various human SULTs are summarised in Table 1. The results of many studies show clearly that the expression of SULTs in humans is carefully regulated with respect to tissue type, development and hormonal influences, supporting proposed functional roles for some of the enzymes. A number of examples are worth considering further: SULTs 1C2 and 1C4 appear to be most highly expressed in fetal tissues,¹³ although RNA dot blots indicated the adult stomach and kidney (SULT1C2) and ovary (SULT1C4) may be sites of expression.^18,19 However the function of these enzymes in humans is not clear. The catecholamine sulfotransferase SULT1A3 is expressed at high levels in fetal liver but hepatic expression is essentially absent in the adult¾here the gastrointestinal tract is the major site¹² which correlates with the dopaminergic function of the gut, where the majority of dopamine sulfate is produced.⁴ The fetal adrenal gland produces large amounts of DHEA sulfate to support placental estrogen biosynthesis, a requirement reflected in the high level expression of the SULT2A1 isoform in the fetal zone of 2nd and 3rd trimester adrenal.^9,20 Another important example involves the estrogen sulfotransferase (SULT1E1) which displays a particularly high affinity (in the low nM range) for its natural substrate 17-estradiol, suggesting an important role for this enzyme in modulating estrogen action.²¹ Indeed, the enzyme is expressed in the endometrium and is exquisitely regulated during the menstrual cycle^22,23 (probably under the primary influence of progesterone^24,25) where it is postulated to moderate estrogenic stimulation of the endometrium around the time of implantation.²³ A further insight into the importance of estrogen sulfation has come from the identification that a number of hydroxylated metabolites of the ubiquitous environmental pollutants polychlorinated biphenyls are extraordinarily potent inhibitors (K_i in the pM range) of SULT1E1,²⁶ suggesting a mechanism whereby these chemicals exert their well-known endocrine disrupting effects.²⁷

A particularly intriguing problem currently occupying a number of investigators in the field is to determine the function of the most recently discovered SULT family, the 4A proteins. SULT4A1 cDNAs have been isolated from three mammalian species so far¾rat, mouse and human¾and the predicted proteins share a remarkable degree of similarity. The mouse and the rat proteins have identical amino acid sequences, and the human has only six (mainly conservative) amino acid changes out of 284. Human SULT4A1 shares no more than 34% amino acid sequence identity with any other member of the family. The SULT4A1 proteins appear to be expressed only in the brain,²⁸ and despite significant effort no natural or xenobiotic substrate (ligand) has yet been identified. Together, these observations strongly suggest a specific and important function for this protein, which may require the application of knockout and/or transgenic technologies to fully understand.

The concept of sulfation as a particularly important metabolic reaction in the developing human fetus is one that has also matured over recent years. Many SULTs are expressed at high levels in the fetus (Table 1), and in fact some appear to be expressed only or primarily in the prenatal period, such as the SULT1C enzymes.^13,18,19 The function of certain SULTs in fetal development is fairly well established, such as the production of DHEA sulfate by the adrenal SULT2A1 enzyme.⁹ However, the role of others is not so clear. SULT1C enzymes are expressed (in sexually dimorphic manner) in adult rodent liver where they seem to be involved in xenobiotic metabolism¾particularly the bioactivation of procarcinogens. This may not be the case in humans, where the enzymes are reputedly involved in thyroid hormone metabolism¾this is a particularly important function in the fetus where appropriate thyroid hormone homeostasis is essential for normal brain development. The human fetus produces very large amounts (compared with the adult) of iodothyronine sulfates (our unpublished work in collaboration with R Hume). The high levels of SULT1A1 in various fetal tissues¹² may provide an important chemical defence function, particularly in the absence from the liver of the other major conjugating enzymes such as UDP-glucuronosyltransferases¹⁵ (although the fetal kidney does express some UDP-glucuronosyltransferase²⁹). These studies are extremely difficult to carry out, however the wide distribution and tight regulation of SULT expression during human development points to a particularly important role for these enzymes in the early stages of life.

STRUCTURE-FUNCTION STUDIES ON SULFOTRANSFERASES

Structural biology is one of the most powerful tools available for helping to understand macromolecular function, and structural approaches have recently revealed some interesting and potentially valuable secrets of the SULTs. The first SULT structure to be solved, by the group of Pedersen and Negishi at NIEHS using X-ray crystallography, was that of mouse SULT1E1.³⁰ This enzyme is unusual in the SULT world in that, in vitro, it exists as a monomer in solution¾most of the enzymes appear to form homodimers. Structures of two other human cytosolic SULTs have since been solved: SULT1A3^31,32 and SULT2A1,³³ as well as the sulfotransferase domain of human heparan sulfate N-deacetylase/N-sulfotransferase³⁴ and the SULT-like insect enzyme retinol dehydratase.³⁵ Figure 1 shows the overall SULT protein fold, as demonstrated by the mouse SULT1E1 crystal structure.

The three cytosolic SULT structures are highly conserved, and a number of key features of the SULT active site and reaction mechanism (reviewed in³⁶) can be deduced from these and other studies (see Figure 2). The amino acids involved in binding the sulfuryl donor PAPS are, as would be expected, highly conserved throughout the SULT family. The PAPS 5'-phosphate binding site (consensus sequence TYPKSGTTW, corresponding to residues 45-53 in SULT1A3) is very similar to the P-loop motif found in many nucleotide binding proteins, and is called the PSB-loop in SULTs.³⁰ The lysine residue within this motif (Lys₄₈ in human SULT1A3 and mouse SULT1E1) is absolutely conserved and is believed to act as a catalytic acid in the reaction, by protonating the phosphate-sulfate bridge oxygen of PAPS thereby enhancing dissociation of the leaving group.³⁷ Residues from various parts of the polypeptide chain form the binding site for the 3'-phosphate of PAPS. Arg₁₃₀ and Ser₁₃₈ (Ser₁₃₈ is absolutely conserved in sulfotransferases) are particularly important, along with the guanidinium group of Arg₂₅₇ and the amide nitrogens of Lys₂₅₈ and Gly₂₅₉ that contribute to a motif highly conserved within the cytosolic SULTs (RKGxxGxWK, residues 257-265 in SULT1A3). The catalytic centre of the enzyme is a histidine residue (His₁₀₈) that acts as a catalytic base, abstracting a proton from the hydroxyl group on the acceptor substrate leaving a nucleophilic oxygen that 'attacks' the sulfur on PAPS. The presence of a histidine residue in this position is an absolutely conserved feature of all SULTs, and mutating it abolishes enzyme activity.³⁷ The reaction intermediate (at least in mouse SULT1E1³⁷) is stabilised by His₁₀₈, Lys₁₀₆ and Lys₄₈.

Of course, a vital feature of the SULT enzyme family is the degree of substrate specificity demonstrated by the individual isoforms. For example, humans have enzymes that are highly selective for catecholamines (SULT1A3), estrogens (SULT1E1) and DHEA (SULT2A1). Conversely the SULT1A1 enzyme appears to be much more promiscuous, able to sulfate a wide variety of xenobiotics with high affinity. It is a challenging problem to determine what controls the substrate specificity of these various enzymes, but this is an important goal from a number of perspectives, including being able to predict the metabolic fate of drugs metabolised by sulfation, or even to assist the design of selective inhibitors. The protein folds of the SULTs whose structures have been solved are essentially superposable, however some subtle, but important differences in the active sites of the enzymes do exist. The human SULT1A3 enzyme provides a fine example: it displays a high selectivity for endogenous and xenobiotic catecholamines (particularly dopamine) not shared by its close relative SULT1A1 even though the two enzymes are more than 93% identical at the amino acid sequence level¾ie only 20 residue differences out of 295. Analysis of these sequence differences³⁸ showed that a number of them occur in a short stretch (143-HRMEKAHPEP-152) immediately C-terminal to a conserved sequence that we now know to form the -6 helix.³¹ Mutation of two amino acids in this stretch of SULT1A3 to their corresponding residues in SULT1A1 (His₁₄₃Tyr and Glu₁₄₆Ala) followed by detailed kinetic analysis of the purified, recombinant proteins clearly demonstrated that Glu₁₄₆ was the major determinant of the specificity of SULT1A3 for dopamine³⁸ (Figure 2). The group of McManus performed similar studies, including the reverse experiment on SULT1A1, arriving at the same conclusion.^39,40 Subsequent molecular modelling experiments based on the X-ray crystal structure of SULT1A3 suggested a molecular basis for this specificity, as the basic ethylamine function in dopamine and similar molecules would be in close proximity to the acidic carboxyl side chain of Glu₁₄₆, and able to form a salt bridge.³¹ The influence of Glu₁₄₆ goes further however, and probably controls the orientation of many compounds in the SULT1A3 active site, explaining the selectivity of SULT1A3 for catechols over corresponding phenols.³¹ The only other major study on SULT selectivity suggests that the specificity of mouse SULT1E1 for 17-estradiol over DHEA arises from a 'gating' phenomenon. Petrotchenko et al⁴¹ demonstrated that Tyr₈₁ and Phe₁₄₂ form a narrow gate that 'allows' 17-estradiol entry to the active site but 'prevents' DHEA (which has a 19-methyl group) from entering. Phe₁₄₂ is absolutely conserved in SULTs, and Tyr₈₁ alone was found to regulate the gate phenomenon.

Clearly, our understanding of the molecular basis for SULT substrate specificity is still in its infancy. The structural tools now available to us will certainly allow major advances to be made in this important area of sulfation biology in the coming years.

INTER-INDIVIDUAL VARIATION IN SULFOTRANSFERASES¾MECHANISMS AND CONSEQUENCES

Ever since studies of human SULT biochemistry began it has been obvious that the activity of these enzymes varies widely in the population. Early work focused on the phenol SULTs (those we now call SULTs 1A1 and 1A3), and was facilitated by the use of platelets as enzyme source. These studies have been thoroughly reviewed elsewhere.^42,43,44 The key questions relating to inter-individual variability in any SULT are, of course: (a) what is the molecular basis?; and (b) what are the physiological consequences? We are considerably further advanced in our ability to answer the former, although several recent molecular epidemiological studies have begun to suggest roles for SULT polymorphisms in disease susceptibility.

SULT1A1 Pharmacogenetics

SULT1A1 is the most widely studied polymorphic SULT, and is significantly due to its broad substrate specificity, wide tissue distribution and involvement in the metabolism and detoxification of many drugs and other xenobiotics as well as in the bioactivation of dietary and environmental procarcinogens.⁴⁵ Biochemical and genetic studies (using twins and other family cohorts) indicated that variation in this 'thermostable' phenol SULT activity was regulated by inheritance,⁴⁶ and also suggested that this variation might be associated with sulfation capacity for drugs such as paracetamol in vivo.^47,48 In addition to variation in SULT1A1 enzyme activity, it was also discovered that the thermal stability of this enzyme activity was under the control of a genetic polymorphism.⁴⁹ Differences in thermal stability can arise from subtle changes in protein structure (eg single amino acid changes), and these studies gave the first clues as to the molecular basis for inter-individual variation in SULT1A1 activity. Development of specific antibodies recognising human SULTs on immunoblot analysis allowed confirmation that, in most tissues at least, the variability in SULT1A1 enzyme activity strongly correlates with the level of cytosolic enzyme protein†.^50,51 The next major advances came with the cloning of human SULT1A1 cDNAs, which provided the first evidence that coding region sequence variant alleles of SULT1A1 existed in the population. There are now eight characterised SULT1A1 cDNA sequences in the GenBank/EMBL sequence databases, which mainly represent the two most common SULT1A1 alleles determined by gene sequencing studies, termed SULT1A1^*1 and SULT1A1^*2^,53 (Table 2). Some of these cDNA sequences contain nucleotide variants that have never been found in gene sequencing studies, which either represent extremely rare SULT1A1 alleles, or (perhaps more likely) have arisen from PCR and/or sequencing errors (GenBank/EMBL accession numbers L19999, U26309 and X84654). Two entries encode the wild-type SULT1A1^*1 allozyme (X78283 and AJ007418) and three encode the common variant allozyme SULT1A1^*2 (L10819, L19955, U09031).

Seminal studies from Weinshilboum's group confirmed that platelet phenol sulfotransferase enzyme activity and thermal stability were related to SULT1A1 genotype. They showed that individuals homozygous for the SULT1A1^*2 allele had only about 15% of the platelet phenol sulfotransferase activity of 1A1^*1/1A1^*2 heterozygotes or 1A1^*1 homozygotes, and also that the thermal stability of enzyme activity was associated with SULT1A1 genotype.^52,53 A subsequent study confirmed that reduced platelet SULT activity is observed in 1A1^*2 homozygotes,⁵⁴ although the effect was not as pronounced with only a 50% reduction in activity apparent. It remains to be conclusively proven how the single amino acid change (Arg₂₁₃His) present in SULT1A1^*2 results in decreased enzyme activity, protein expression and thermal stability. Experiments with the recombinant proteins have, in the main, failed to show significant differences in kinetic properties between SULTs 1A1^*1 and 1A1^*2, although the thermal stability of the SULT1A1^*2 allozyme appears considerably reduced compared to the wild-type enzyme.^53,55,56 Recent work from this laboratory using recombinant enzymes expressed and purified from E. coli suggests that the 1A1^*2 allozyme may be a more catalytically efficient enzyme, and it is certainly considerably less susceptible to the partial substrate inhibition phenomenon displayed by SULT1A1^*1 (Tabrett CA and Coughtrie MWH, unpublished work). However, the 1A1^*2 allozyme protein appears to have a shorter biological half-life than the wild-type, and preliminary data suggest that it is more readily degraded via the proteasome pathway.⁵⁷ This would explain the various observations¾reduced activity, reduced protein expression and thermal instability¾that have been reported. Examination of the SULT crystal structure does not provide an obvious mechanistic explanation; amino acid 213 is on the surface of the molecule distant from the active site and from the proposed dimerisation domain.⁵⁸

The fact that SULT1A1 is: (a) important for xenobiotic detoxification, thyroid hormone metabolism⁵⁹ and the bioactivation of procarcinogens,⁴⁵ and (b) subject to a common functional polymorphism has inspired a number of investigators to carry out molecular epidemiological studies in an attempt to associate SULT1A1 genotype with various allegedly chemical-associated pathologies, including colorectal, breast and prostate cancer. Predictably, such studies have produced conflicting results. It is also interesting that a significant age-related effect on SULT1A1 genotype has been observed, whereby the frequency of the SULT1A1^*1 allele was higher in the older age-groups.⁶⁰ This observation raises the possibility that the genotype responsible for a high sulfation activity phenotype may provide some protection against long-term cell/tissue damage from xenobiotic and/or endogenous chemicals, and as such would support a major role for SULT1A1 in detoxification. Although this finding requires to be confirmed in a larger and more tightly defined ageing population, it does illustrate the importance of careful age matching for any case-control studies involving genotyping.

Frame et al⁶¹ reported that low platelet phenol SULT activity (attributed to SULT1A1) was more prevalent in colorectal cancer patients. These results support data indicating that SULT1A1^*1 homozygosity is associated with reduced risk of colorectal cancer.⁶² However, a large study of breast cancer patients showed no overall effect of SULT1A1 genotype on risk of developing the disease, although these authors did demonstrate that genotype may be associated with age of onset of breast cancer.⁶³ In contrast, investigation of another breast cancer patient cohort showed increased risk associated with SULT1A1^*1 genotype when combined with intake of well-done meat, a significant source of many heterocyclic amines⁶⁴ that are bioactivated by human SULTs including SULT1A1.^45,65 To complicate matters, however, this same study⁶⁴ found that overall risk of breast cancer was associated with the SULT1A1^*2 allele. A small study of prostate cancer failed to find any association with SULT1A1 genotype.⁶⁶ Clearly, these diseases are multi-factorial and it would be surprising to find very strong associations with SULT1A1 genotype. Only through large-scale studies of diseases where sulfation may be important, and accounting for specific environmental exposures, will we gain greater insight into the function of SULTs and sulfation in humans.

In common with other drug metabolising enzymes,^67,68 there appear to be inter-ethnic/inter-racial differences in the incidence of the various SULT1A1 alleles (Table 3). African-Americans appear to have a particularly high frequency of the 1A1^*3 allele, associated with reduced 1A1^*1 allele frequency¾1A1^*2 allele frequency is not significantly different to Caucasian populations. In contrast, the 1A1^*1 allele is present at significantly higher frequencies in oriental populations (Chinese, Japanese) compared with either the Caucasian or African-American populations studies so far. The significance of these differences in terms of sulfation function is not yet clear, and further studies in this area are certainly required. However, these observations suggest that different ethnic/racial populations may display different capacities for sulfation via SULT1A1.

SEQUENCE AND FUNCTIONAL VARIATION IN OTHER HUMAN SULFOTRANSFERASES

SULT Family 1

Coding region single nucleotide polymorphisms resulting in amino acid changes (ie non-synonymous cSNPs) have been identified in a number of Family 1 SULTs. Five cSNP variants of SULT1A2 are known, from either cDNA or gene sequencing studies,^53,69,70 (Table 2), and experiments in recombinant systems suggest that the protein produced by the common variant SULT1A1^*2 (Ile₇Thr, Asn₂₃₅Thr) has a substantially higher K_m for the substrate 4-nitrophenol.^53,69 The functional significance of this is not clear, and investigating it is complicated: (a) by the possibility that a significant portion of SULT1A2 RNA is incorrectly spliced and therefore protein production is hampered⁷¹ (indeed we have seen no evidence for SULT1A2 protein by western blot analysis in various human tissues¹³); and (b) by the observation that the most common variant alleles of SULT1A1 and SULT1A2 (ie the ^*2 alleles) are in strong positive linkage disequilibrium.^53,70,72 No cSNP polymorphisms in SULT1A3 have been published to date,⁷³ and in fact this gene seems remarkably devoid of sequence variation between individuals.

The SULT1B1 genes from 48 Japanese have been sequenced and a single cSNP identified (Glu₂₀₄Asp), although no allele frequency for this variant was reported.⁷³ A number of non-coding region SNPs were also identified in this study.

Sequencing of the human SULT1C2 gene from different individuals has revealed four cSNPs (Table 2), and recombinant allozymes representing three of these displayed, reduced enzyme activity compared to the wild-type allozyme.⁷⁴ A common 30bp insertion/deletion variant within intron three of SULT1C2 also exists,⁷⁴ and there is a rare SULT1C2 transcript variant where an additional exon (exon 3b) is spliced into the mRNA molecule.⁷⁵ The consequences for the individual of possessing these variant alleles and/or expressing the transcript variant are unknown, although the SULT1C2 enzyme is expressed at high levels in fetal tissues and has been implicated in the bioactivation of aromatic amines,^19,76 and also in the metabolism of iodothyronines.^59,77 A single cSNP in SULT1C4 (Asp₅Glu) was identified in a sequencing study of many SULT genes in 48 Japanese individuals, although no allele frequency or allozyme function data were reported.⁷³

The expression and activity of SULT1E1 varies widely in the human population,^23,78 although it is not known whether this is under genetic control. No evidence for gender-specific regulation in liver was found,⁷⁸ although the enzyme is expressed in female-specific tissues, including the endometrium. It is possible that the variability in SULT1E1 expression results from different chemical influences, since progesterone, other hormones and alcohol are known to influence expression levels in vitro and/or in vivo.^24,25,78 The only published study on SULT1E1 nucleotide sequence variants reported a number of non-coding region variants (5'- and 3'-flanking regions, introns) but no cSNPs.⁷³ However, data reported by the Pharmacogenetics Knowledge Base (http://www.pharmgkb.org/data/mayo/SULT1E1a_files/frame.htm) indicate the presence of very rare (allele frequency 0.008) SULT1E1 cSNPs in the Caucasian (Ala₃₂Val and Pro₂₅₃His) and African-American (Asp₂₂Tyr) populations. Again, no functional characterisation of the resulting variant allozymes has been reported.

SULT Families 2 and 4

Circulating DHEA sulfate levels vary substantially between individuals, and it is suggested that this variation is, at least in part, inherited.⁷⁹ It is reasonable to hypothesise that inter-individual variation in the expression and/or activity of SULT2A1 might therefore contribute to these differences in DHEA sulfate levels. Indeed, DHEA SULT activity in human liver cytosols appears to follow a bimodal distribution, and a strong correlation between enzyme activity and protein expression exists.⁸⁰ Analysis of SULT2A1 cDNA sequences isolated by a number of laboratories shows the presence of the presumed wild-type sequence (SULT2A1^*1, GenBank/EMBL accession numbers U08024, U08025 and X84816¾consistent with the two SULT2A1 gene sequences U13056-U13061 and L36191-L36196) and two apparent cSNPs (Thr₉₀Ser, L20000 and X70222; Leu₁₅₉Val, S43859, L02337). However, extensive gene sequencing studies⁸¹ failed to identify the variant alleles represented by these cDNAs. These authors did report two cSNPs (Met₅₇Thr, Glu₁₈₆Val) with allele frequencies around 3% which have been assigned the preliminary designation ^*2 and ^*3, respectively,⁸² (Table 2). The SULT2A1^*2 and ^*3 allozymes showed decreased enzyme activity when expressed in COS cells.⁸¹ A further three cSNPs in SULT2A1 (Ala₆₃Pro, Lys₂₂₇Glu and Ala₂₆₁Thr) were originally reported by the Pharmacogenetics Knowledge Base (http://www.pharmgkb.org/data/mayo/SULT2A1a_files/frame.htm) and have recentlybeen published⁸³ (Table 2). They have only been found in African-American populations; Ala₂₆₁Thr is common, with an allele frequency of 13%. This may be significant as inter-ethnic differences in SULT2A1 activity were reported.⁸⁰ Interestingly, the most common of the variants found in African-American subjects (Ala₂₆₁Thr) influences the formation of SULT2A1 dimers in vitro,⁸³ as it occurs within the conserved dimerisation motif.⁵⁸ No non-synonymous cSNPs in SULT2A1 or SULT2B1 were found in the Japanese cohort.⁷³

In one study, a large number of SNPs were found in the SULT4A1 gene (whose function remains to be determined), although all 42 occurred in non-coding regions.⁷³ This is perhaps surprising, given the extremely highly conserved nature of the SULT4A1 protein from mouse to human. Another SULT with an important endogenous function, SULT1A3, shows an extremely low level of variation and one might have expected the same pattern for SULT4A1.

FUTURE DIRECTIONS IN SULFOTRANSFERASE RESEARCH

Significant advances have been made over recent years in our understanding of the SULT enzyme family, and of the sulfation system as a whole, including (but not restricted to) those outlined here. There is no reason to doubt this trend will continue, and it may be driven, at least in part, by the needs of the Pharmaceutical Industry. The fact that many adverse drug reactions are directly related to genetic and/or environmental effects on the major cytochromes P450 (particularly CYP2D6 and the CYP3A family)⁸⁴ has led many in the industry, as well as the regulatory authorities, to re-evaluate drug discovery and development priorities away from compounds whose primary route of metabolism is via the CYP system.⁸⁵ The consequence of this is that many drugs coming to market in the future will be metabolised primarily by other pathways, in particular the conjugating enzymes such as UDP-glucuronosyltransferases and SULTs. This is already beginning to happen and the example of the anti-diabetic drug troglitazone illustrates the point. Inhibition of bile salt transport by troglitazone sulfate has been implicated in the severe idiosyncratic hepatoxicity of this drug⁸⁶ which led to its withdrawal from the US market in 2000, only 3 years after its launch. It will therefore be necessary to know much more about the biology and genetics of these enzymes in both human and experimental animal species if we are to be able to predict adverse reactions involving compounds metabolised by conjugation. This will require the application of a range of experimental approaches, including functional genomics, nucleic acid and protein array technologies, high-resolution/high-sensitivity analytical techniques such as mass spectrometry and NMR spectroscopy, structural biology, in vivo metabolic studies and transgenic/knockout experiments. Studies of in vivo sulfation in humans are thin on the ground¾lack of good, safe, specific probe substrates is one major stumbling block¾but such analyses are of key importance in assessing the functional consequences of inter-individual variation in SULTs. Much useful information on the functions of cytochromes P450 have come from such studies, and there is a desperate need for them to be conducted on sulfation. Hopefully this will be an area where significant advances can be made in the future.

The example of troglitazone also highlights another important area for future research¾the transport of substrates and products of the sulfation reaction into and out of cells. There has been an explosion in research into small molecule transport in recent years (see reviews^87,88,89) and the major families of transport proteins (MRPs, OATPs and BSEP) are important components of the sulfation system. It will be important to understand the substrate specificities of these multiple transporters for sulfate conjugates and SULT substrates, and also to explore the interactions of endogenous transporter substrates such as bile salts, steroid and thyroid hormones with sulfate conjugates generated during xenobiotic metabolism.

Finally, it is worth highlighting one very recent landmark advance in the field: the generation of the first SULT knockout mice.⁹⁰ Using standard targeting vector methodology, these investigators generated mice deficient in estrogen SULT (SULT1E1). The main phenotype of the knockout mice was observed in males, where age-dependent Leydig cell hypertrophy/hyperplasia coupled with seminiferous, tubule damage occurred. These lesions resulted in abnormal sperm function (reduced motility) and consequently the older male mice produced smaller litters. In young SULT1E1 knockout mice, however, a reduction in fertility was also observed although this was associated with the female animals. These elegant studies certainly provide solid evidence that estrogen sulfation is important for (male) reproductive function in mice. However, as with all animal experiments, the relevance to humans must be carefully assessed and clearly demonstrated. Although there is some evidence (RT-PCR data only) that SULT1E1 is expressed in the human testis⁹¹ there are fundamental differences between mice and humans in the expression profile of this important enzyme. For example, hepatic expression is sexually dimorphic in mice⁹² but not in humans,⁷⁸ in humans the enzyme is expressed at much higher levels in fetal than adult tissues,¹³ and a major and critical site of expression in adult humans is the endometrium.²³ It is certain this will be the first of many knockout/transgenic experiments involving SULTs, and much useful information will undoubtedly come from such studies.

So, there is certainly much of interest in the SULT field currently, as there will be in the foreseeable future, to appeal to the curious scientist and/or clinician. The highlights presented here are, of necessity, a personal selection, although hopefully they cover the more pertinent aspects of this fascinating system relating to human biology. These and other studies directed towards understanding the function of sulfation and the SULTs will undoubtedly reveal important roles for the enzymes that are not yet obvious, but that we will look back on from a more enlightened future.

Acknowledgements

I am grateful to all the members of my laboratory (past and present), and to numerous valued collaborators, who have contributed to the work and ideas described here. I am particularly indebted to Dr Rebecca Raftogianis (Fox Chase Cancer Center, Philadelphia, USA) for her critical reading of the manuscript and for helpful suggestions regarding nomenclature, and to Professor Jyrki Taskinen (University of Helsinki, Finland) for producing the model upon which Figure 2 is based. Current research in the laboratory is supported by the Medical Research Council, the Commission of the European Communities (QLG3-CT-2000-00930), Tenovus Scotland, the Scottish Office Chief Scientist Office and the Human Drug Conjugation Consortium.

DUALITY OF INTEREST

*Raftogianis RB et al, submitted for publication. In particular, the new nomenclature for the SULT1C enzymes may cause some confusion. The human SULT1C2 referred to here was called SULT1C1 or SULT1C sulfotransferase 1 in the original descriptions,^18,19,75 and the human SULT1C4 referred to here was originally called SULT1C2 or SULT1C sulfotransferase.^19,75

†This may not be the case for liver, where the presence of other SULT isoforms such as 1B1 may interfere with measurement of enzyme activity with non-selective substrates such as 4-nitrophenol (our unpublished work).

1 Robbins PW, Lipmann F. Isolation and identification of active sulfate. J Biol Chem 1957; 229: 837-851.

2 Klaassen CD, Boles JW. The importance of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in the regulation of sulfation. FASEB J 1997; 11: 404-418.

3 Dousa MK, Tyce GM. Free and conjugated plasma catecholamines, DOPA and 3-O-methyldopa in humans and various animal species. Proc Soc Exp Biol Med 1988; 188: 427-434.

4 Eisenhofer G, Coughtrie MWH, Goldstein DS. Dopamine sulphate: an enigma resolved. Clin Exp Pharmacol Physiol 1999; 26: S41-S53.

5 Ogura K, Kajita J, Narihata H, Watabe T, Ozawa S, Nagata K et al. Cloning and sequence analysis of a rat liver cDNA encoding hydroxysteroid sulfotranferase. Bio chem Biophys Res Commun 1989; 165: 168-174.

6 Ogura K, Kajita J, Narihata H, Watabe T, Ozawa S, Nagata K et al. cDNA cloning of the hydroxysteroid sulfotransferase STa sharing a strong homology in amino acid sequence with the senescence marker protein SMP-2 in rat livers. Biochem Biophys Res Commun 1990; 166: 1494-1500.

7 Watabe T, Ogura K, Satsukawa M, Okuda H, Hiratsuka A. Molecular cloning and functions of rat liver hydroxysteroid sulfotransferases catalysing covalent binding of carcinogenic polycyclic arylmethanols to DNA. Chem Biol Interact 1994; 92: 87-105.

8 Otterness DM, Wieben ED, Wood TC, Watson RWG, Madden BJ, McCormick DJ et al. Human liver dehydroepiandrosterone sulfotransferase: molecular cloning and expression of cDNA. Mol Pharmacol 1992; 41: 865-872. MEDLINE

9 Barker EV, Hume R, Hallas A, Coughtrie MWH. Dehydroepiandrosterone sulfotransferase in the developing human fetus¾quantitative biochemical and immunological characterization of the hepatic, renal, and adrenal enzymes. Endocrinology 1994; 134: 982-989.

10 Hume R, Barker EV, Coughtrie MWH. Differential expression and immunohistochemical localization of the phenol and hydroxysteroid sulfotransferase enzyme families in the developing lung. Histochem Cell Biol 1996; 105: 147-152.

11 Hume R, Coughtrie MWH. Phenolsulfotransferase¾localization in kidney during human embryonic and fetal development. Histochem J 1994; 26: 850-855.

12 Richard K, Hume R, Kaptein E, Stanley EL, Visser TJ, Coughtrie MWH. Sulfation of thyroid hormone and dopamine during human development¾ontogeny of phenol sulfotransferases and arylsulfatase in liver, lung and brain. J Clin Endocrinol Metab 2001; 86: 2734-2742.

13 Stanley EL. Expression and function of iodothyronine metabolising enzymes during human placental and fetal development. PhD Thesis University of Dundee, 2001.

14 Hakkola J, Pelkonen O, Pasanen M, Raunio H. Xenobiotic-metabolizing cytochrome P450 enzymes in the human feto-placental unit: role in intrauterine toxicity. Crit Rev Toxicol 1998; 28: 35-72.

15 Coughtrie MWH, Burchell B, Leakey JEA, Hume R. The inadequacy of perinatal glucuronidation¾immunoblot analysis of the developmental expression of individual UDP-glucuronosyltransferase isoenzymes in rat and human-liver microsomes. Mol Pharmacol 1988; 34: 729-735.

16 Varin L, Marsolais F, Richard M, Rouleau M. Biochemistry and molecular biology of plant sulfotransferases. FASEB J 1997; 11: 517-525.

17 Weinshilboum RM, Otterness DM, Aksoy IA, Wood TC, Her C, Raftogianis RB. Sulfotransferase molecular biology: cDNAs and genes. FASEB J 1997; 11: 3-14. MEDLINE

18 Her C, Kaur GP, Athwal RS, Weinshilboum RM. Human sulfotransferase SULT1C1: cDNA cloning, tissue-specific expression and chromosomal localization. Genomics 1997; 41: 467-470.

19 Sakakibara Y, Yanagishita M, Katafuchi J, Ringer DP, Takami Y, Nakayama T et al. Molecular cloning, expression, and characterization of novel human SULT1C sulfotransferases that catalyze sulfonation of N-hydroxy-2-acetylaminofluorene. J Biol Chem 1998; 273: 33929-33935.

20 Parker CR, Falany CN, Stockard CR, Stankovic AK, Grizzle WE. Immunohistochemical localization of dehydroepiandrosterone sulfotransferase in human fetal tissues. J Clin Endocrinol Metab 1994; 78: 234-236.

21 Song WC, Melner MH. Editorial: steroid transformation enzymes as critical regulators of steroid action in vivo. Endocrinology 2000; 141: 1587-1589.

22 Buirchell BJ, Hähnel R. Metabolism of estradiol-17b in human endometrium during the menstrual cycle. J Steroid Biochem 1975; 6: 1489-1494.

23 Rubin GL, Harrold AJ, Mills JA, Falany CN, Coughtrie MWH. Regulation of sulfotransferase expression in the endometrium during the menstrual cycle, by oral contraceptives and during early pregnancy. Mol Human Reprod 1999; 5: 995-1002.

24 Tseng L, Liu HC. Stimulation of arylsulfotransferase activity by progestins in human endometrium in vitro. J Clin Endocrinol Metab 1981; 53: 418-421.

25 Falany JL, Falany CN. Regulation of estrogen sulfotransferase in human endometrial adenocarcinoma cells by progesterone. Endocrinology 1996; 137: 1395-1401.

26 Kester MHA, Bulduk S, Tibboel D, Meinl W, Glatt H, Falany CN et al. Potent inhibition of estrogen sulfotransferase by hydroxylated PCB metabolites: a novel pathway explaining the estrogenic activity of PCBs. Endocrinology 2000; 141: 1897-1900. MEDLINE

27 Safe SH. Polychlorinated biphenyls (PCBs): environmental impact, biochemical and toxic responses, and implications for risk assessment. Crit Rev Toxicol 1994; 24: 87-149. MEDLINE

28 Falany CN, Xie XW, Wang J, Ferrer J, Falany JL. Molecular cloning and expression of novel sulphotransferase-like cDNAs from human and rat brain. Biochem J 2000; 346: 857-864.

29 Hume R, Coughtrie MWH, Burchell B. Differential localization of UDP-glucuronosyltransferase in kidney during human embryonic and fetal development. Arch Toxicol 1995; 69: 242-247.

30 Kakuta Y, Pedersen LG, Carter CW, Negishi M, Pedersen LC. Crystal structure of estrogen sulphotransferase. Nature Struct Biol 1997; 4: 904-908. MEDLINE

31 Dajani R, Cleasby A, Neu M, Wonacott AJ, Jhoti H, Hood AM et al. X-ray crystal structure of human dopamine sulfotransferase, SULT1A3: molecular modelling and QSAR analysis demonstrate a molecular basis for sulfotransferase substrate specificity. J Biol Chem 1999; 274: 37862-37868. Article MEDLINE

32 Bidwell LM, McManus ME, Gaedigk A, Kakuta Y, Negishi M, Pedersen L et al. Crystal structure of human catecholamine sulfotransferase. J Mol Biol 1999; 293: 521-530. Article MEDLINE

33 Pedersen LC, Petrotchenko EV, Negishi M. Crystal structure of SULT2A3, human hydroxysteroid sulfotransferase. FEBS Lett 2000; 475: 61-64. Article MEDLINE

34 Kakuta Y, Sueyoshi T, Negishi M, Pedersen LC. Crystal structure of the sulfotransferase domain of human heparan sulfate N-deacetylase/N-sulfotransferase 1. J Biol Chem 1999; 274: 10673-10676. Article MEDLINE

35 Pakhomova S, Kobayashi M, Buck J, Newcomer ME. A helical lid converts a sulfotransferase to a dehydratase. Nat Struct Biol 2001; 8: 447-451.

36 Yoshinari K, Petrotchenko EV, Pedersen LC, Negishi M. Crystal structure-based studies of cytosolic sulfotransferase. J Biochem Molec Toxicol 2001; 15: 67-75.

37 Kakuta Y, Petrotchenko EV, Pedersen LC, Negishi M. The sulfuryl transfer mechanism: crystal structure of a vanadate complex of estrogen sulfotransferase and mutational analysis. J Biol Chem 1998; 273: 27325-27330. Article MEDLINE

38 Dajani R, Hood AM, Coughtrie MWH. A single amino acid, Glu₁₄₆, governs the substrate specificity of a human dopamine sulfotransferase, SULT1A3. Mol Pharmacol 1998; 54: 942-948.

39 Brix LA, Barnett AC, Duggleby RG, Leggett B, McManus ME. Analysis of the substrate specificity of human sulfotransferases SULT1A1 and SULT1A3: site-directed mutagenesis and kinetic studies. Biochemistry 1999; 38: 10474-10479.

40 Brix LA, Duggleby RG, Gaedigk A, McManus ME. Structural characterization of human aryl sulphotransferases. Biochem J 1999; 337: 337-343.

41 Petrotchenko EV, Doerflein ME, Kakuta Y, Pedersen LC, Negishi M. Substrate gating confers steroid specificity to estrogen sulfotransferase. J Biol Chem 1999; 274: 30019-30022. Article MEDLINE

42 Weinshilboum R. Phenol sulfotransferase inheritance. Cell Mol Neurobiol 1988; 8: 27-33.

43 Weinshilboum R. Sulfotransferase pharmacogenetics. Pharmac Ther 1990; 45: 93-107.

44 Weinshilboum R, Aksoy I. Sulfation pharmacogenetics in humans. Chem Biol Interact 1994; 92: 233-246.

45 Glatt H, Engelke CEH, Pabel U, Teubner W, Jones AL, Coughtrie MWH et al. Sulfotransferases: genetics and role in toxicology. Toxicol Lett 2000; 112: 341-348.

46 Price RA, Spielman RS, Lucena AL, van Loon JA, Baidak BL, Weinshilboum RM. Genetic polymorphism for human platelet thermostable phenol sulfotransferase (TS PST) activity. Genetics 1989; 122: 905-914.

47 Reiter C, Weinshilboum R. Platelet phenol sulfotransferase activity: correlation with sulfate conjugation of acetaminophen. Clin Pharmacol Ther 1982; 32: 612-621.

48 Bonham-Carter SM, Rein G, Glover V, Sandler M, Caldwell J. Human platelet phenol sulphotransferase M and P: substrate specificities and correlation with in vivo sulphoconjugation of paracetamol and salicylamide. Br J Clin Pharmacol 1983; 15: 323-330.

49 van Loon JA, Weinshilboum RM. Human platelet phenol sulfotransferase: familial variation in thermal stability of the TS form. Biochem Genet 1984; 22: 997-1014.

50 Jones AL, Roberts RC, Coughtrie MWH. The human phenolsulfotransferase polymorphism is determined by the level of expression of the enzyme protein. Biochem J 1993; 296: 287-290.

51 Stanley EL, Hume R, Visser TJ, Coughtrie MWH. Differential expression of sulfotransferase enzymes involved in thyroid hormone metabolism during human placental development. J Clin Endocrinol Metab 2001; 86: 5944-5955.

52 Raftogianis RB, Wood TC, Otterness DM, van Loon JA, Weinshilboum RM. Phenol sulfotransferase pharmacogenetics in humans: association of common SULT1A1 alleles with TS PST phenotype. Biochem Biophys Res Commun 1997; 239: 298-304. Article MEDLINE

53 Raftogianis RB, Wood TC, Weinshilboum RM. Human phenol sulfotransferases SULT1A2 and SULT1A1. Genetic polymorphisms, allozyme properties, and human liver genotype-phenotype correlations. Biochem Pharmacol 1999; 58: 605-616. Article MEDLINE

54 Nowell S, Ambrosone CB, Ozawa S, MacLeod SL, Mrackova G, Williams S et al. Relationship of phenol sulfotransferase activity (SULT1A1) genotype to sulfotransferase phenotype in platelet cytosol. Pharmacogenetics 2000; 10: 789-797.

55 Ozawa S, Shimizu M, Katoh T, Miyajima A, Ohno Y, Matsumoto Y et al. Sulfating-activity and stability of cDNA-expressed allozymes of human phenol sulfotransferase, ST1A3^*1 (213Arg) and ST1A3^*2 (213His), both of which exist in Japanese as well as Caucasians. J Biochem 1999; 126: 271-277.

56 Li X, Clemens DL, Cole JR, Anderson RJ. Characterization of human liver thermostable phenol sulfotransferase (SULT1A1) allozymes with 3,3',5'-triiodothyronine as the substrate. J Endocrinol 2001; 171: 525-532.

57 Raftogianis RB. Human sulfotransferases and cellular response to estrogens and antiestrogens. Drug Metab Rev 2001; 33: 10.

58 Petrotchenko EV, Pedersen LC, Borchers CH, Tomer KB, Negishi M. The dimerization motif of cytosolic sulfotransferases. FEBS Lett 2001; 490: 39-43.

59 Kester MHA, Kaptein E, Roest TJ, van Dijk CH, Tibboel D, Meinl W et al. Characterization of human iodothyronine sulfotransferases. J Clin Endocrinol Metab 1999; 84: 1357-1364.

60 Coughtrie MWH, Gilissen RAHJ, Shek B, Strange RC, Fryer AA, Jones PW et al. Phenol sulphotransferase SULT1A1 polymorphism: molecular diagnosis and allele frequencies in Caucasian and African populations. Biochem J 1999; 337: 45-49.

61 Frame LT, Gatlin TL, Kadlubar FF, Lang NP. Metabolic differences and their impact on human disease¾Sulfotransferase and colorectal cancer. Environ Toxicol Pharmacol 1997; 4: 277-281.

62 Bamber DE, Fryer AA, Strange RC, Elder JB, Deakin M, Rajagopal R et al. Phenol sulphotransferase SULT1A1^*1 genotype is associated with reduced risk of colorectal cancer. Pharmacogenetics 2001; 11: 679-685.

63 Seth P, Lunetta KL, Bell DW, Gray H, Nasser SM, Rhei E et al. Phenol sulfotransferases: hormonal regulation, polymorphism, and age of onset of breast cancer. Cancer Res 2000; 60: 6859-6863. MEDLINE

64 Zheng W, Xie DW, Cerhan JR, Sellers TA, Wen WQ, Folsom AR. Sulfotransferase 1A1 polymorphism, endogenous estrogen exposure, well-done meat intake, and breast cancer risk. Cancer Epidem Biomar 2001; 10: 89-94.

65 Chou HC, Lang NP, Kadlubar FF. Metabolic activation of the N-hydroxy derivative of the carcinogen 4-aminobiphenyl by human tissue sulfotransferases. Carcinogenesis 1995; 16: 413-417.

66 Steiner M, Bastian M, Schulz WA, Pulte T, Franke KH, Rohring A et al. Phenol sulphotransferase SULT1A1 polymorphism in prostate cancer: lack of association. Arch Toxicol 2000; 74: 222-225.

67 Kalow W, Bertilsson L. Interethnic factors affecting drug response. Advanc Drug Res 1994; 23: 1-53.

68 Nebert DW, Menon AG. Pharmacogenomics, ethnicity, and susceptibility genes. Pharmacogenomics J 2001; 1: 19-22.

69 Zhu X, Veronese ME, Iocco P, McManus ME. cDNA cloning and expression of a new form of human aryl sulfotransferase. Int J Biochem Cell Biol 1996; 28: 565-571.

70 Engelke CEH, Meinl W, Boeing H, Glatt H. Association between functional genetic polymorphisms of human sulfotransferases 1A1 and 1A2. Pharmacogenetics 2000; 10: 163-169.

71 Dooley TP, Haldeman-Cahill R, Joiner J, Wilborn TW. Expression profiling of human sulfotransferase and sulfatase gene superfamilies in epithelial tissues and cultured cells. Biochem Biophys Res Commun 2000; 277: 236-245.

72 Carlini EJ, Raftogianis RB, Wood TC, Jin F, Zheng W, Rebbeck TR et al. Sulfation pharmacogenetics: SULT1A1 and SULT1A2 allele frequencies in Caucasian, Chinese and African-American subjects. Pharmacogenetics 2001; 11: 57-68.

73 Iida A, Sekine A, Saito S, Kitamura Y, Kitamoto T, Osawa S et al. Catalog of 320 single nucleotide polymorphisms (SNPs) in 20 quinone oxidoreductase and sulfotransferase genes. J Hum Genet 2001; 46: 225-240. Article MEDLINE

74 Freimuth RR, Eckloff B, Wieben ED, Weinshilboum RM. Human sulfotransferase SULT1C1 pharmacogenetics: gene resequencing and functional genomic studies. Pharmacogenetics 2001; 11: 747-756.

75 Freimuth RR, Raftogianis RB, Wood TC, Moon E, Kim UJ, Xu J et al. Human sulfotransferases SULT1C1 and SULT1C2: cDNA characterization, gene cloning, and chromosomal localization. Genomics 2000; 65: 157-165.

76 Yoshinari K, Nagata K, Shimada M, Yamazoe Y. Molecular characterization of ST1C1-related human sulfotransferase. Carcinogenesis 1998; 19: 951-953.

77 Li XY, Clemens DL, Anderson RJ. Sulfation of iodothyronines by human sulfotransferase 1C1 (SULT1C1). Biochem Pharmacol 2000; 60: 1713-1716.

78 Song W-C, Qian YM, Li AP. Estrogen sulfotransferase expression in the human liver: marked interindividual variation and lack of gender specificity. J Pharmacol Exp Ther 1998; 284: 1197-1202.

79 Rotter JI, Wong FL, Lifrak ET, Parker LN. A genetic component to the variation of dehydroepiandrosterone sulfate. Metabolism 1985; 34: 731-736. MEDLINE

80 Aksoy IA, Sochorová V, Weinshilboum RM. Human liver dehydroepiandrosterone sulfotransferase¾nature and extent of individual variation. Clin Pharmacol Ther 1993; 54: 498-506. MEDLINE

81 Wood TC, Her C, Aksoy I, Otterness DM, Weinshilboum RM. Human dehydroepiandrosterone sulfotransferase pharmacogenetics: quantitative western analysis and gene sequence polymorphisms. J Steroid Biochem Mol Biol 1996; 59: 467-478. Article MEDLINE

82 Nagata K, Yamazoe Y. Pharmacogenetics of sulfotransferase. Annu Rev Pharmacol Toxicol 2000; 40: 159-176.

83 Thomae BA, Eckloff BW, Freimuth RR, Wieben ED, Weinshilboum RM. Human sulfotransferase SULT2A1 pharmacogenetics: genotype-to-phenotype studies. Pharmacogenomics J 2002; 2: 48-56.

84 Ingelman-Sundberg M. Implications of polymorphic cytochrome P450-dependent drug metabolism for drug development. Drug Metab Dispos 2001; 29: 570-573. MEDLINE

85 Hodgson J. ADMET¾turning chemicals into drugs. Nat Biotechnol 2001; 19: 722-726.

86 Funk C, Ponelle C, Scheuermann G, Pantze M. Cholestatic potential of troglitazone as a possible factor contributing to troglitazone-induced hepatotoxicity: In vivo and in vitro interaction at the canalicular bile salt export pump (Bsep) in the rat. Mol Pharmacol 2001; 59: 627-635.

87 Keppler D, Kamisako T, Leier I, Cui Y, Nies AT, Tsujii H et al. Localization, substrate specificity, and drug resistance conferred by conjugate export pumps of the MRP family. Adv Enzyme Regul 2000; 40: 339-349.

88 Suzuki H, Sugiyama Y. Transport of drugs across the hepatic sinusoidal membrane: sinusoidal drug influx and efflux in the liver. Semin Liver Dis 2000; 20: 251-263.

89 Kullak-Ublick GA, Stieger B, Hagenbuch B, Meier PJ. Hepatic transport of bile salts. Semin Liver Dis 2000; 20: 273-292.

90 Qian YM, Sun XJ, Tong MH, Li XP, Richa J, Song WC. Targeted disruption of the mouse estrogen sulfotransferase gene reveals a role of estrogen metabolism in intracrine and paracrine estrogen regulation. Endocrinology 2001; 142: 5342-5350.

91 Song W-C, Qian Y, Sun X, Negishi M. Cellular localization and regulation of expression of testicular estrogen sulfotransferase. Endocrinology 1997; 138: 5006-5012.

92 Borthwick EB, Burchell A, Coughtrie MWH. Differential expression of hepatic estrogen, phenol and dehydroepiandrosterone sulfotransferases in genetically-obese diabetic (ob/ob) male and female mice. J Endocrinol 1995; 144: 31-37.

93 Meloche CA, Falany CN. Expression of SULT2B1b in hormonally regulated human tissues. Drug Metab Rev 2001; 33: 230.

94 Geese WJ, Raftogianis RB. Biochemical characterization and tissue distribution of human SULT2B1. Biochem Biophys Res Commun 2001; 288: 280-289.

95 Javitt NB, Lee YC, Shimizu C, Fuda H, Strott CA. Cholesterol and hydroxycholesterol sulfotransferases: identification, distinction from dehydroepiandrosterone sulfotransferase, and differential tissue expression. Endocrinology 2001; 142: 2978-2984.

96 Ozawa S, Tang Y-M, Yamazoe Y, Kato R, Lang NP, Kadlubar FF. Genetic polymorphism in human liver phenol sulfotransferases involved in the bioactivation of N-hydroxy derivatives of carcinogenic arylamines and heterocyclic amines. Chem Biol Interact 1998; 109: 237-248.

Box 1 Sulfotransferase Reaction and Co-substrate Synthesis

Figure 1 Structure of mouse SULT1E1. The co-ordinates for sulfotransferase 1E1 from mouse crystallised in the presence of PAP and substrate 17-estradiol were obtained from the Protein Data Bank (http://www.rcsb.org/pdb/, entry 1AQU). A representation of the structure of a SULT1E1 monomer is shown, with -helical sections displayed as red cylinders and -sheets as light blue arrows. The figure was created using WebLab Viewer Lite (Accelrys, San Diego, USA).

Figure 2 Active site model of human SULT1A3. To construct the active site model, the co-ordinates of mouse SULT1E1 (PDB entry 1AQU) and the partial human SULT1A3 structure³¹ were used. Molecule A of 1AQU was used as a template, and the SULT1A3 structure was super-imposed as the -carbons of residues 42-45, 45-50, 52-58, 104-107, 136-147 and 166-170. Side chains of the SULT1E1 component covering PAP and 17 -estradiol (residues 225-264) were mutated to their corresponding residues in SULT1A3, and dopamine was docked into the active site based on the position of estradiol in 1AQU, so that the 3-OH is on the estradiol phenolic OH and the 4-OH is towards Tyr₂₄₀. The figure was created using WebLab Viewer Lite (Accelrys, San Diego, USA).

Box 2 The Sulfotransferase Enzyme Superfamily

Table 1 Properties of the human cytosolic sulfotransferases

Table 2 Human sulfotransferase allozymes

Table 3 Ethnic differences in SULT1A1 allele frequencies