DRUG METABOLISM REVIEWS

Vol. 36, Nos. 3 & 4, pp. 823-843, 2004

Sulfonation in Pharmacology and Toxicology*

Frederick C. Kauffman*
Laboratory for Cellular and Biochemical Toxicology, Ernest Mario School
of Pharmacy, Rutgers, The State University of New Jersey,
Piscataway, New Jersey, USA

ABSTRACT
Sulfonation has a major function in modulating the biological activities of a wide
number of endogenous and foreign chemicals, including: drugs, toxic chemicals,
hormones, and neurotransmitters. The activation as well as inactivation of many
xenobiotics and endogenous compounds occurs via sulfonation. The process is
catalyzed by members of the cytosolic sulfotransferase (SULT) superfamily consisting
of at least ten functional genes in humans. The reaction in intact cells may be reversed
by arylsulafatase present in the endoplasmic reticulum. Under physiological
conditions, sulfonation is regulated, in part, by the supply of the co-substrate/donor
molecule 3'-phosphadensoine-5-phosphosulfate (PAPS), and transport mechanisms by
which sulfonated conjugates enter and leave cells. Variation in the response of
individuals to certain drugs and toxic chemicals may be related to genetic
polymorphisms documented to occur in each of the above pathways. Sulfonation
has a major function in regulating the endocrine status of an individual by modulating
the receptor activity of estrogens and androgens, steroid biosynthesis, and the
metabolism of catecholamines and iodothyronines Sulfonation is a key reaction in the
body's defense against injurious chemicals and may have a major function during
early development since SULTs are highly expressed in the human fetus. As with

"Supported in part by NIEHS Center Grant No. ES05052.

*Correspondence: Frederick C. Kauffman, Laboratory for Cellular and Biochemical Toxicology,
Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ
08854, USA; E-mail: kauffma@rci.rutgers.edu.
823
DOI: I0.1081/DMR-200033496
Copyright 2004 by Marcel Dekker, Inc.

0360-2532 (Print); 1097-9883 (Online)

www.dekker.com

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KaufTman

many Phase I and Phase II reactions, sulfonation may also serve as the terminal step in
activating certain dietary and environmental agents to very reactive toxic
intermediates implicated in carcinogenesis.
Key Words: Sulfonation; Sulfation; Sulfotransferase; SULT; Sulfatase; PAPS.

INTRODUCTION
Sulfonation of low molecular weight compounds catalyzed by members of the
cytosolic sulfotransferase multigene family (SULT) is an important determinant of the
pharmacology and toxicology of a vast array of endogeneous and foreign chemicals
(Coughtrie, 2002; Strott, 2002). This pathway involves the transfer of a sulfonate group
(S0''~) from the universal donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to an
appropriate substrate. Sulfonated conjugates are often incorrectly referred to as
"sulfates" because the transfer of an SO^ ~ group to a hydroxyl acceptor creates an
SO"* ester. Various components of the sulfonation pathway are summarized in Fig. 1.
The sulfonation system resides primarily in cytosol but involves interaction with

The Sulfonation System

ATP.SO,

-SO4"

/
^ P S Kinase

Sulfotransferase:SULT
- ROSO3"

Cytosol

Endoplasmic Reticulum

Sulfatase:ARSc

Figure 1. Scheme depicting interactions between various factors that influence the net formation
and transport of sulfonate esters from intact cells. Availability of the obligatory cofactory, PAPS, is
present at relatively low concentrations and may limit the synthesis of sulfonate conjugates
catalyzed by various SULTs. PAPS is formed via a single bifunctional enzyme that contains both
ATP sulfurylase and APS activities. Inorganic sulfate and two molecules of ATP are required for
each molecule of PAPS syntheisized. Sulfonate ester formation may also be reversed via the
hydrolytic enzyme arylsulfatas-c present in the endolplasmic reticulum. Sulfonate conjugates leave
and enter cells via specific specific transporters including mdr and organic acid transport molecules.
Inter-individual variation in each of the above enzymatic activities imposed by genetic and/or
environmental events may limit the process of sulfonation intact cells and organs.

Sulfonation in Pharmacology and Toxicology

825

arylsulfates-c (ARSc) in the endoplasmic reticulum and specific transport molecules

localized in the plasma membrane. Both organic acid transport molecules (OAT) (Buist
et al., 2003) and multidrug resistant proteins (mdrs) (Chu et al., 2004) have been
implicated in coupling sulfonate conjugate formation to transport from and into cells.
Sulfonate conjugates may also allosterically regulate mrp-mediated transport of other
conjugates such as glucuronides (Chu et al., 2004). PAPS is synthesized via ATP
sulfurylase and APS kinase which reside within a single bifunctional enzyme of
approximately 56 kDa (Lyle et al., 1994). Formation of sulfonated conjugates from
various substrates is catalyzed by cytosolic sulfotransferases (SULTs). Arylsulfatases
present in intact cells have the capacity to reverse the process of sulfonation in cells
and thus influence the process of net sulfonation (Coughtrie et al., 1998; Kauffman
et al., 1991; Tan and Pang, 2001). Under physiological conditions, each of these
components; availability of substrate and PAPS, as well as activities of both synthetic
SULTs and hydrolytic sulfatases, influence net availability of sulfonated conjugates.
The importance of sulfonation in modulating the function of many endogenous
compounds, including a large number of neurotransmitters and hormones as well as an
enormous number of xenobiotics in humans, is well established. Several excellent
reviews have appeared recently that consider progress made in understanding the
biological function of sulfonation and the regulation of expression of SULT isoforms in
specific tissues during critical periods of development (Coughtrie, 2002; Glatt et al.,
2001; Strott, 2002). The object of this review is to highlight some important historical
aspects of the field and briefly review work on the sulfonation of xenobiotics and
endobiotics of particular interest to pharmacologists and toxicologists.
The process of sulfonation in biology was discovered in the late 19th century by
Eugen Baumann (1876), who isolated and characterized phenol sulfate from the urine
of a patient given phenol as an antiseptic. The enzymatic basis for the process was
discovered 80 years later when Lipmann's group discovered "active sulfate" in their
pioneering studies on the metabolism of sulfanilamide (Robbins and Lippman, 1957).
This compound, which is the sulfuryl donor for most sulfonation reactions, is now
know to be 3-phosphoadenosyl- 5'-phosphosulfate (PAPS) (Klaassen and Bowles,
1997). Understanding the important role of sulfonation in the pharmacology and
toxicology of endogenous and foreign chemicals has advanced greatly since the
discovery of "active sulfate," and in many ways has paralleled research with other
families of drug metabolizing enzymes including the cytochrome P450s and UDP
glucuronosyl transferases. Research underlying the great progress that has been made in
the past few years, due in large measure to the application of techniques in molecular
biology and modeling, is the subject of a number of recent reviews, (Coughtrie, 2002;
Weinshilboum et al., 1997). In light of Herbert Remmer's life time contributions to the
field of drug metabolism and his early work on both oxidative and conjugative
reactions (Bock et al., 1973; Remmer et al., 1975), he would be gratified by the
exciting work on sulfonation that has followed his pioneering studies.
Like many drug metabolizing enzymes, cytosolic SULTs compose a large
superfamily of genes. Full length cDNA for more than 50 mammalian and avian
species have been cloned and sequenced (Coughtrie, 2002; Nagata and Yamazoe,
2000). Cytosolic sulfotransferases described to date have been classified into five
families that share less than 40% similarity with each other based on their amino acid

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KaufTman

sequences. SULTl and SULT2 families are the largest and are responsible for
sulfonating the greatest number of endogenous and foreign, compounds. There are
currently 11 known isoforms of human SULT enzymes representing the SULTl,
SULT2, and SULT4 families (Coughtrie, 2002). These are products of 10 genes, with
alternate splicing occurring with the first exon of the SULT2B1 gene as reviewed in
(Weinshilboum et al., 1997). The expression of SULTs is carefully regulated in terms
of tissue type, development, and hormonal regulation. Some of the properties of known
human SULT isoforms with regard to major sites of expression and specificities for
endogenous and xenobiotic substrates are summarized in recent reviews by Coughtrie
and his colleagues (Coughtrie, 2002; Coughtrie et al., 1998). Despite considerable
research, endobiotic and xenobiotic ligands for a number of SULT isoforms remain to
be identified. An interesting example here is the failure to identify substrates for a
highly conserved protein (SULT4A1) identified from the expressed sequence database
that appears to be expressed only in mammalian brain (Falany et al., 2000; Liyou et al.,
2003; Sakakibara et al., 2002). This protein is prominent in a number of brain
structures including the cerebral cortex, cerebellum, pituitary, and brainstem of rats and
humans; however to date, no endogenous nor xenobiotic substrate has yet been
identified for SULT4A1.

PHARMACOGENTICS
Inter-individual variation in expression of SULT isoforms that have pharmacological and toxicological significance in humans are well established [for reviews see
(Coughtrie, 2002; Weinshilboum and Aksoy, 1994)]. Considerable information exists
concerning the molecular basis underlying variation in SULT activities, and a number
of molecular epidemiological studies linking SULT polymorphisms to disease
susceptibility have appeared (Bamber et al., 2001; Seth et al., 2000; Zheng et al.,
2001). Pioneering studies carried out by Weinshilboum and his colleagues showed that
platelet phenol sulfotransferase activity and thermal stability were related to SULTIAl
genotype (Haenen et al., 1991; Weinshilboum and Aksoy, 1994). The availability of a
simple colorimetric assay, and the presence of thermal stable and thermal labile forms
of phenol sulfotransferase in platelets, an easily accessible tissue, opened the possibility
of initiating studies into the heritability of biochemically distinct forms of SULT. Early
studies employing biochemical measurements showed that genetically determined
variation in the thermal stability of phenol sulfotransferase in platelets correlated with
individual differences in sulfonation of acetaminophen after oral administration (Reiter
and Weinshilboum, 1982). Using 4-nitrophenol as a substrate, Raftogianis et al. (1997)
observed more than a 50-fold variation in the activity of phenol sulfotransferase from
905 subjects. This enzyme, now known as SULTIAl, is a "broad spectrum"
sulfotransferase involved in the metabolism and detoxification of many drugs and other
foreign chemicals as well as the bioactivation of many dietary and environmental
procarcinogens (Coughtrie and Johnston, 2001; Glatt et al., 2000).
Given its historical backgound and broad substrate specificity, SULTIAl is the
most widely studied polymorphic sulfotransferase. Subsequent to the earlier
biochemical studies, molecular cloning of human SULTIAl cDNAs indicated that
variation in thermal stability and activity are related to genetically determined variants

Sulfonation in Pharmacology and Toxicology

827

in the coding region of this enzyme in humans (Coughtrie, 2002; Weinshilboum and
Otterness, 1994). The two most common SULTIAl alleles, termed SULT1A1*1 and
SULT1A1*2, determined by gene sequencing studies involve a single amino acid
change at position 213. The allelic variant that possesses an arginine at position 213
(SULT1A1*1) is more thermostabile than the variant containing histadine at this
position (SULTIA 1*2) when using p-nitrophenol as substrate. Pioneering work by
Weinshilboum and his colleagues showed that platelet enzyme activity and thermal
stability were related to SULTIAl genotype. Individuals with the SULTIAl*2
genotype had significantly lower platelet activity than 1A1*1/1A1*2 heterozygotes or
1A1*1 homozygotes (Raftogianis et al., 1997). The reduced activity noted in
SULTIAl*2 genotypes is likely due to alterations in amounts of expressed enzyme
since kinetic properties of recombinant SULTIAl isoforms failed to demonstrate
reduced activity of the 1A1*2 allozyme (Li et al., 2001; Tabrett and Coughtrie, 2003).
A reduced biological half-life due to enhanced proteosomal degradation has been
suggested as a possible mechanism accounting for low amounts of the SULTIAl*2
allozyme (Coughtrie, 2002).
The occurrence of a common functional polymorphism in SULTIAl has
stimulated a number of molecular epidemiological studies attempting to link individual
variation in SULTIAl activity with certain pathologies notably breast (Nowell et al.,
2002a; Saintot et al., 2003; Seth et al., 2000; Zheng et al., 2001), colon (Bamber et al.,
2001; Liang et al., 2003), prostate (Nowell et al., 2004), and lung (Liang et al., 2003)
cancers. Although these studies have produced confiicting results, they are valuable in
identifying associations that may occur between certain physiological factors, life
styles, and SULTIAl genotypes. For example, a significant increase in the frequency
of the wild type allele, SULTlAl*l, was noted in older individuals in a small
population-based study suggesting that the high sulfonation phenotype provided
protection against long term tissue damage arising from exposures to endogenous
chemicals or xenobiotcs with aging (Coughtrie et al., 1999). In contrast, several case
control studies concerning the onset of various cancers mentioned above suggest an
association between the high activity SULT1A]*1 allele and heightened risk of these
diseases in human populations. There is at least one study indicating that homozygosity
for the SULT]A1*2 allele slightly reduced the risk for colorectal cancer (Nowell
et al., 2002b).
A potential link between high activity and SULTIAl and increased risk from
certain dietary constituents has been suggested. A large number of environmental
mutagens and carcinogens, such as heterocyclic amines contained in well done meat,
are activated by sulfotransferases (Glatt, 2000) . The risk of early-onset breast cancer as
well as the occurrence of other tumors may be increased in individuals having higher
amounts the SULTIAl allele compared to controls (Seth et al., 2000). Several studies
suggesting that variations in SULTIAl alleles contribute to the risk of breast (Zheng
et al., 2001) and prostate (Nowell et al., 2004) cancer implied that risk to these cancers
was increased by the consumption of well done meats. Findings such as these, as well
as those of other studies, emphasize the importance of considering associations between
host/environmental factors and SULTIAl genotypes in relation to ultimate biological outcomes.
The functional significance of polymorphisms in other members of the SULTl
family have been given much less attention than those noted in SULTIAl; however.

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Kauffman

evidence is emerging that variation in these genes also contribute to pathophysiology in

humans. Sequencing of DNA samples for 60 African Americans and 60 Caucasian
American subjects identified one non-synonymous SNP (Lys234Arg) only in African
American subjects (Thomae et al., 2003). Heterologous expression of this variant in
COS cells indicated decreased sulfonation of dopamine compared to wild type enzyme
expressed in the same system. A significant decrease in immunoreactive protein
compared to that of the wild type sequence was noted. Kinetic properties of the two
recombinant proteins did not differ. The authors provide data that this single amino
acid change leads to accelerated degradation through a proteosome-mediated process
(Thomae et al., 2003).
Estrogen sulfotransferase (SULTIEI) has the lowest Km values for estrogens of
the 11 known human SULT enzymes. A number of studies suggest that inter-individual
variation in the activity of this isoform is associated with the pathophysiology of
hormone-dependent diseases such as breast cancer. Using the same cohort of African
American and Caucasian Americans described above, Weinshilboum and his colleagues
identified three non-synonymous SNPs that were associated with altered levels of
expression and kinetic properties of sequences expressed heterologously in COS-1 cells
(Adjei et al., 2003). Such findings lend further credence to the idea that genetically
determined variation in SULTIEI catalyzed sulfonation of estrogens contributes to the
pathophysiology of estrogen-dependent diseases as well as the biotransformation of
estrogens administered pharmacologically. Application of DNA sequencing and
analysis of the SULT2A1 gene and expression constructs derived from the same
cohort above identified three functional SNPs in DNA from African American subjects
(Thomae et al., 2001). The most common of these non-synonymous SNPs occurred in
the dimerization motive (Petrotchenko et al., 2001) that influenced the dimerization of
SULT2A1 in vitro.
PAPS is synthesized from ATP and inorganic sulfate by two isoforms of PAPS
synthetase (PAPSSl and PAPSS2) (Besset et al., 2000; Xu et al., 2000). A rare
polymorphism (GLU532) in the PAPSSl identified in DNA from an African American
subject was associated with a five-fold higher Km for inorganic sulfate compared to the
wild-type allozyme (Xu et al., 2003). It is not known if this genetic variation in
PAPSSl is associated with decreased PAPS synthesis and altered sulfonate conjugation
in vivo.

SULFONATION OF ENDOGENOUS AND

FOREIGN CHEMICALS
Sulfotransferses from each of the major subfamilies of this group of enzymes are
involved in metabolism of broad classes of foreign and endogenous compounds. There
are several recent excellent reviews summarizing these reactions. An earlier review by
Miller and Surh (1994) summarized the key function that sulfonation has in the
activation of a wide array of chemical carcinogens. There are also several recent
reviews by Glatt and his colleagues summarizing tbe activation of chemical
carcinogens by specific members of the SULT family (Glatt et al., 2000; Thomae
et al., 2001). An extensive review on the enzymatic aspects of members of the SULTl
family has also recently appeared as have reviews on the involvement of SULTs in the

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Sulfonation in Pharmacology and Toxicology

Table 1.
Substrate
Etidogettous chemicals
Catecholamines

Selected substrates for mammalian sulfotransferases.

SULT isoform

SULT lAl, SULT 1A3

Iodothyronines

SULTIAl

Ascorbic acid
Vitamin D

SULTIAl
SULT2A1

Cholesterol

SULT2Blb

Estrogens

SULTIEI, lAl, 1A2

Dhydroepiandrosterone

SULT2A1

Androgens
Neurosteroids

SULTIAl
SULT2A1

Bile acids

SULT2A1

Xenobiotics
Drugs
Acetaminophen
Apomorphine
Butesonide
Ethinylestradiol
Minoxidil

SULTIAl
SULTIAl, 1A2,
1A3, lEl
SULT2A1
SULTIAl
SULTIAl, lCl

Tamoxifen

SULTIAl

Dietary constituents
Curcumin
Flavonoids

SULTIAl, IA3
SULTIAl, 1A3, lCl

Epicatachin

SULTIAl, 1A3

Reference

(Buu etal., 1981;

Eisenhofer et al., 1999;
Taskinen et al., 2003;
Thomae et al., 2003)
(Kester et al., 2003;
Li and Anderson, 1999;
Li et al., 2000; Rubin et al., 1999;
Visser, 1994; Visser et al., 1998)
(Tolbert, 1985)
(Axelson, 1985;
Echchgadda et al., 2004)
(Strott and Higashi, 2003;
Yanai et al., 2004)
(Adjei and Weinshilboum, 2002;
Mancini et al., 1992;
Purinton and Wood, 2000;
Qian et al., 2001)
(Shimada et al., 2001;
Sugahara et al., 2003)
(Chang et al., 2004)
(Cascio et al., 2000;
Krueger and Papadopoulos, 1992;
Markowski et al., 2001)
(Frye and Lacey, 1999;
Kitada et al., 2003;
Palmer and Bolt, 1971;
Shen et al., 2000;
Song et al., 2001)

(Reiter and Weinshilboum, 1982)

(LeWitt, 2004;
Thomas and Coughtrie, 2003)
(Meloche et al., 2002)
(Chu et al., 2004)
(Dooley, 1999;
Meisheri et al., 1993)
(Chen et al, 2002;
Glatt et al., 1998)
(Ireson et al., 2002)
(Mesia-Vela and Kauffman, 2003;
Pai et al., 2001;
Peterson et al., 1998)
(Mesia-Vela and Kauffman, 2003)
(continued)

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Kauffman
Table 1.

Substrate
Toxic chemicals
Pro-carcinogens
Aliphatic and
benzylic alcohols
Alkenylbenzenes
Aromatic amines
and amides
Heterocyclic
aromatic amines
Polynuclear aromatic
hydrocarbons
Others
Perflorocarboxylic acids
Manganese

Continued.

SULT isoform

SULT2A1
SULTIAl
SULTIAl, SULTIC

SULTIAl, 1A2, IA3

SULTIAl

SULTIAl
SULTIAl

Reference
(Miller and Surh, 1994)
(Duffel etal., 2001;
Glatt et al., 2000)
(Boberg et al., 1983)
(Glatt and Meinl, 2004;
King and Phillips, 1968;
Sakakibara et al., 1998)
(Buonarati et al., 1990;
Sugimura, 2002)
(Tiemersma et al., 2004;
Watabe etal., 1982)
(Witzmann et al., 1996)
(Ranasinghe et al., 2000)

biotransformation of endogeneous compounds (Coughtrie, 2002; Strott, 2002).

Members of the SULT2A family are primarily associated with the metabolism of
hydroxysteroids (Thomae et al., 2001; Weinshilboum and Otterness, 1994). Selected
examples of the function of sulfonation in the biotransformation of endogenous
compounds and xenobiotics are summarized in Table 1 and briefly discussed below.

Endogenous Chemicals
Sulfonation has been recognized as a pathway for catecholamine inactivation in
man and animals for at least 3 decades (Buu et al., 1981; Roth and Rivett, 1982), and it
has been estimated that as much as 10% of the metabolism of dopamine and
norepinephrine in brain may be inactivated by this pathway (Rivett et al., 1982;
Whittemore and Roth, 1985). Most catechois studied are substrates for SULT isoforms
lAl, 1A2, 1A3, and lBl (Taskinen et al., 2003). A functional genetic polymorphysim
recently reported for human SULT 1A3 has been associated with accelerated
degradation via a proteosomal-mediated process (Thomae et al., 2003). Authors of
this report raised the possibility that such changes may be related to inherited
alterations in catecholamine sulfonation in humans. Dopamine sulfate exists at much
higher concentrations in human plasma than dopamine and appears to arise mainly
from dietary biogneic amines and sulfonation of dopamine produced in the myenteric
plexus of the gastroinitestinal tract via SULTl A3 which is expressed in large amounts
in the gastrointestinal tract (Eisenhofer et al., 1999). It has been suggested that more
than 75% of dopamine sulfate present in the body is produced via this pathway which
serves as a "gut-blood" barrier for dietary biogenic amines and dopamine produced in
the myenteric plexus (Kester et al., 2002).
There is a growing awareness of the importance of sulfonation during human fetal
development. Many SULTs that are produced in high amounts in human fetal tissues

Sulfonation in Pharmacology and Toxicology

831

have been linked to important developmental processes (Coughtrie, 2002). For example, production of DHEA sulfate via SULT2A1 by the fetal adrenal is established as
a critical step in providing substrate for estrogen biosynthesis by the placenta during
pregnancy (Barker et al., 1994). Thyroid hormone bioavailability in the human fetus is
regulated, in part, by enzymatic deiodination and reversible sulfonation of
iodothyronines (Hume et al., 2001). Sulfonation of 3,3'-diiodothyronine was found to
correlate with SULTIAl in a wide range of fetal tissues suggesting that this isoform is
primarily responsible for sulfonation of this hormone in fetal tissues (Richard et al.,
2001). SULTIAl may serve an important defense mechanism in the fetus since other
metabolic pathways are very low or absent in fetal tissues, and the human fetus
produces very high amounts of iodothyronine sulfates (Coughtrie, 2002).
Cholesterol sulfate and DHEA sulfate are the two most abundant sterol sulfonates
in the human circulation (Strott and Higashi, 2003). Their concentrations overlap and
range between 2 to 6 [xM in blood; however, while blood levels of cholesterol sulfate
remain relatively constant throughout life, levels of DHEA sulfate peak at puberty and
decline with age (Orentreich et al., 1984). Much is known about the physiological
function of the former in contrast to DHEA sulfate which, in large, remains a mystery.
In contrast, considerable information exists concerning the role of cholesterol sulfate as
a regulatory molecule in a variety of processes, e.g., keratinocyte differentiation,
epidermal and platelet cell adhesion, sperm capacitation, blood clotting, and fibrinolysis
(Strott and Higashi, 2003). Both compounds are sulfonated by members of the SULT2
family, which are primarily involved in the conjugation of neutral steroids and sterols.
DHEA is considered as the primary substrate for SULTIAl, which is also referred to
as DHEA sulfotransferase or hydroxysulfotransferase (Nagata and Yamazoe, 2000).
SULT2B has been further divided into two isoforms derived from the same gene
(SULTIBI) differing in structure and substrate specificity; SULT2Bla and SULT2Blb
(Her et al., 1998). SULT2Blb acitively sulfonates cholesterol while SULT2Bla
sulfonates pregnenolone but not cholesterol (Fuda et al., 2002; Strott and Higashi,
2003). The recent finding that SULT2Blb is expressed in human platelets (Yanai et al.,
2004) enhances the opportunity to explore potential polymorphisms in this gene and
relationships to altered physiological processes regulated by cholesterol sulfate.
The term neurosteroids designates steroids that are newly synthesized from
cholesterol or other precursors in the nervous system, and are still present in substantial
amounts after removal of peripheral steroidogenic organs (Mensah-Nyagan et al.,
1999). Initial steps in the synthesis of neurosteroids from cholesterol involves of
number of cytochrome P450s localized in brain (Hojo et al., 2004; Shibuya et al.,
2003). Sulfonation in brain is particularly important because a variety of neurotransmitter systems including GABA (Baulieu, 1998; Sullivan and Moenter, 2003),
cholinergic (Rhodes et al., 1997), glutaminergic (Flood et al., 1999) and a-opioid
(Monnet et al., 1995) receptors are modulated by both free and sulfonated neurosteroids, often in opposing ways. For example, pregnenolone is a barbiturate-like agonist, whereas its sulfonated conjugates acts as a picrotoxin-like antagonist (Krueger and
Papadopoulos, 1992; Melchior and Allen, 1992). Dehydroepiandrosterone sulfate enhances acetylcholine release from the hippocampus while the unconjugated form does
not (Rhodes et al., 1996). Cellular action of both pregnenolone and DHEA sulfonates
has been associated with enhanced cognitive performance in mammalian and avian
species, (Meyer et al., 2002; Migues et al., 2002).

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Specific SULT isoforms have been identified in brain; however, their localization
in neuronal cellular elements including glia and neurons remains largely undefined.
Unpublished work in our own laboratory indicates that SULTIAl is a major SULT
isoform in rat brain and is localized primarily in neurons, which agrees with earlier
immunohistochemical studies employing polyclonal antibodies to phenol sulftransferase
indicating that this enzyme is found in neurons of rat brain (Zou et al., 1990). This
isoform has been cloned from human hippocampal samples (Zhu et al., 1993). Activity
of SULTIAl is highest in the rat striatum suggesting that it has a primary role in
sulfonating dopamine in contrast to SULTl A3 which is believed to be the major SULT
isoform involved in dopamine sulfonation in human brain. Evidence for the presence of
other isoforms of SULT in brain is limited. Epxeriments using very high concentrations
of DHEA to test for the presence of SULTIAl (Aldred and Waring, 1999) likely
reflect SULTIAl, which is also capable of sulfonating high concentrations of DHEA.
The earliest report of the presence of a novel cDNA reflecting the brain specific
isoform SULT4A was cloned from rat and human brain (Falany et al., 2000).

Drugs
The diversity of drugs and other xenobiotics biotransformed by sulfonation reactions
has been recognized for many years (Mulder, 1981), and recent studies have greatly
expanded the range of compounds recognized as substrates for these reactions (Mulder and
Jakoby, 1990; Weinshilboum and Otterness, 1994). Examples of recent progress in this
field are illustrated by work on apomorphine (Thomas and Coughtrie, 2003) and
budesonide (Meloche et al., 2002). Apomorphine is a potent dopaminergic agonist used in
the treatment of Parkinson's disease (Hely et al., 2000) and more recently to treat erectile
dysfunction (Kalsi and Kell, 2004). Knowledge that sulfonation is the major route of
metabolism of apomorphine in humans (LeWitt, 2004) led to a study exploring the
metabolism of this drug via seven recombinant isoforms of human SULT (Thomas and
Coughtrie, 2003) SULT lAl, 1A2, 1A3, and lEl, all sulfonated apomorphine to some
extent. By correlating sulfonation of apomorphine in a bank of 27 human liver cytosols
toward SULT activity of either 4-nitrophenol (SULTIAl) or 17b-estradiol, the authors
showed that SULTIAl is the primary isoform responsible sulfonating apomorphine in
human liver. Further studies are required to determine if polymorphisms common in
SULTIAl are associated with variations in drug efficacy and toxicity in humans.
Budesonide is a synthetic glucocorticoid used to treat asthma, allergic reactions,
and infiammatory bowel disease (O'Connell, 2003). This compound is also being
explored in animal models as a novel chemopreventive agent against pulmonary tumors
(Estensen et al., 2004). The drug is distributed as a mixture of two epimers, 22R and
22S, which are metabolized to stereo-specific hydroxylated metabolites by CYP3A4
(Jonsson et al., 1995). In a study of the ability of seven recombinant human SULTs
only SULT2A1 was found capable of sulfonating butesonide (Meloche et al., 2002).
Epimeric forms of the drug also differed in respect to serving as a substrate for
SULT2A1 with the 22 R epimer being 3.5 fold faster than of the 22S epimer.

Dietary Constituents
Many natural and synthetic dietary chemicals are known to inhibit sulfotransferases
including SULTIAl, Suit 1 A3, SULTIEI (Coughtrie and Johnston, 2001). Many of

Sulfonation in Pharmacology and Toxicology

833

these inhibitors are polyphenols such as quereetin (Mesia-Vela and Kauffman, 2003;
Walle et al., 1995), ingredients in red wine (Jones et al., 1995), and tea and coffee
(Coughtrie et al., 1998). Tests with recombinant SULTl isofoms indicated that SULTs
lAl, 1A2, and 1A3 are inhibited to varying degrees by a wide range of dietary
polyphenols; however, the most potent inhibitors found in this class of chemicals were
epicatechin gallate, epigallocatechin, and gallocatechin gallate (Coughtrie and Johnston,
2001). Further kinetic experiments in this same study indicated that inhibitory potency
toward each of the three isoforms varied considerably with SULTIAl being the most
sensitive. Ki values for epicatechin gallate and epigallocatechin gallate were 64 nm and
42 nm, respectively. Further studies to determine the possibility that compounds such
as the gallates in tea and coffee modulate variants of SULTIAl in human subjects in
vivo and the consequences of such effects would be valuable.

Toxic Chemicals
It is well established that sulfotransferases bioactivate a host of chemicals, many of
which are dietary constituents, to reactive intermediates that are implicated in
carcinogenesis. Much of our understanding of the involvement of reactive sulfuric acid
esters in chemical carcinogenesis stems from the pioneering work of James and
Elizabeth Miller (Miller, 1970). Examples of chemicals that are converted to DNA- and
protein adducting species by sulfonation include benzylic alcohols of polycyclic
aromatic hydrocarbons (PAHs), estragole, safrole, as well as various hydoroxyarylamines, arylhydroxamic acids formed from heterocyclic amines found in cooked meat
and fish (Miller and Surh, 1994). A principle pathway for chemical carcinogenesis is
the formation of toxic and very reactive sulfuric acid esters that undergo heterocyclic
cleavage to generate sulfate ions and potent electrophiles that combine.avidly with
nucleophilic groups in cellular DNA and proteins. Much recent work in toxicology has
been devoted to defining the activation of various pro-carcinogens by specific SULT
isoforms (Glatt and Meinl, 2004; Glatt et al., 1995; Sakakibara et al., 1998). A recent
study of nitrofen, that had been used as a herbicide in Germany until its carcinogenic
and teratogenic activities were detected in rodents, illustrates the principle of using
genetically manipulated strains of Salmonella typhimuium to evaluate the role of
specific SULT isoforms in mutagenagenesis (Glatt and Meinl, 2004).
Further appreciation for the importance of sulfonation in toxicology comes from
the finding that a number of hydroxylated metabolites of polychlorinated biphenyls are
extremely potent inhibitors of SULTIEI with Ki's in the picomolar range (Kester et al.,
2000). Since SULTIEI is the primary enzyme responsible for inactivation of estrogens
in humans, the authors suggest that the endocrine disrupting effects of these ubiquitous
environmental pollutants occur via the high capacity of their hydroxylated metabolites
to inhibit estrogen sulfonation via SULTIAl.

FUTURE DIRECTIONS
A major objective of this review was to emphasize the importance of sulfonation in
the biotransformation of a large array of small molecules that are the focus of
pharmacology and toxicology. This process modulates the activities of many endogenous
molecules including steroid hormones and neurotransmitters, and may have a special

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Kauffman

function during fetal and early postnatal life including biotransformation of many
endogenous and foreign chemicals. It will be important to gain further understanding of
mechanisms regulating the expression of various elements of the sulfonation system and
the physiological consequences of alterations in this system during early life.
Evidence collected over the last decade suggest that sulfonation is critical to
regulating the actions of steroids within the central nervous system. Important questions
concerning mechanisms regulating the expression of various elements of the sulfonation
system in brain and other tissues remain to be answered. Some elements involved in
regulating expression of cytochrome P450 enzymes may also function in transcriptionally inducing cytosolic sulfotranferases. For example, the vitamin D receptor
mediating nuclear signaling, known to induce cytochrome P450 expression also appears
to be involved along with the farnesoid X nuclear receptor in stimulating endogenous
SULT2A1 expression (Echchgadda et al., 2004; Song et al., 2001). Progress is being
made in understanding the regulation of this isofrom (e.g., Runge-Morris et al., 1999);
however there is little known concerning mechanisms regulating the expression of other
SULT isoforms.
Finally, progress in understanding interactions between the different components of
the sulfonation system in various organs and tissues is emerging. Recent work
employing mRNA expression and immunohistochemistry has localized steroid sulfatase
and various organic acid transport proteins in biopsy samples of human temporal lobe
(Steckelbroeck et al., 2004). It is tempting to speculate that these components function
together with de novo biosynthesis of DHEA-sulfonate and other 3-3-hydroxy steroids
to regulate levels of these steroids at critical sites in the brain.

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