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Departments of Obstetrics and Gynecology (K.R., R.H., E.L.S.), Child Health (R.H.), and Molecular and Cellular Pathology (R.H., E.L.S., M.W.H.C.), University of Dundee, Ninewells Hospital and Medical School, Dundee, Scotland, United Kingdom DD1 9SY; and Department of Internal Medicine, Erasmus University Medical School (E.K., T.J.V.), 3015 GD Rotterdam, The Netherlands
Address all correspondence and requests for reprints to: Dr. M. W. H. Coughtrie, Department of Molecular and Cellular Pathology, University of Dundee, Level 7, Ninewells Hospital, Dundee, Scotland, United Kingdom DD1 9SY. E-mail: m.w.h.coughtrie{at}dundee.ac.uk
Abstract
Sulfation is an important mechanism for regulating the biological activity of numerous hormones and neurotransmitters in man. Here we have investigated the ontogeny of sulfotransferases (SULT) and sulfatase (ARS) involved in the metabolism of thyroid hormone and dopamine. SULT1A1 enzyme activity was lower in postnatal liver and lung than in fetal tissues. Hepatic SULT1A3 (dopamine) was expressed at high levels early in development, but decreased substantially in late fetal/early neonatal liver and was essentially absent from the adult liver. In lung, significant SULT1A3 activity was observed in the fetus, but neonatal levels were considerably lower. In brain, the highest activity was observed in the choroid plexus for SULT1A1, with low and widespread activity for both SULT1A1 and SULT1A3 in other brain regions. SULT activity with 3,3'-diiodothyronine (3,3'-T2) as substrate was measured in all tissues and correlated significantly with SULT1A1 activity (4-nitrophenol), suggesting that SULT1A1 is primarily responsible for the sulfation of this iodothyronine. The developmental expression of SULT1A3 and SULT1A1 in liver and brain was confirmed by immunoblot, and immunohistochemistry of developing liver showed substantial expression of these proteins in hemopoietic cells in fetal liver. We also detected low activity for the hydrolysis of 3,3'-T2 sulfate by ARS, although there was less distinction between fetal and neonatal samples than with SULT activities. We have therefore shown that the developing fetus has substantial sulfation capacity. Sulfation may therefore play a major role in the homeostasis of hormones and other endogenous compounds as well as in detoxification in the fetus, particularly as other conjugating enzyme systems, such as the UDP-glucuronosyltransferases, are not expressed at significant levels until the neonatal period.
THE DEVELOPING human faces numerous toxic insults from endogenous chemicals and xenobiotics, both in utero and in the postnatal period. Of the major xenobiotic metabolizing enzyme families, the cytochromes P450 are fairly well developed in the fetus (1, 2); however, many of the enzyme systems that make up the major human adult chemical defense mechanism (i.e. the phase 2 or conjugating enzymes) are poorly developed until after birth (e.g. UDP-glucuronosyltransferases) (3, 4), thus compounding the vulnerability of the human fetus. The principal exception to this is sulfation, which appears to be a major mechanism for protection against chemical damage during human development as well as playing a key role in, for example, steroid hormone biosynthesis, catecholamine metabolism, and thyroid hormone homeostasis.
Sulfation is catalyzed by members of the sulfotransferase (SULT) enzyme family, comprising at least 11 members in humans, which transfer a sulfuryl moiety from the universal donor molecule 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to a wide range of acceptor substrates (5, 6, 7). Human SULT enzymes can be subdivided, based on amino acid sequence identity and enzymatic function, into phenol SULT (SULT1) and steroid SULT (SULT2) families, where the SULT1 family comprises enzymes metabolizing phenolic xenobiotics and iodothyronines (SULT1A1) and catecholamines (SULT1A3). A new family with predominant expression in the brain, SULT4A, has recently been described, although its function remains unknown (8, 9). Sulfation normally results in a reduction in biological activity relative to the parent molecule, and sulfate conjugates may be excreted from the body or serve as circulating or intracellular stores (or as metabolic intermediates) from which the free compound can be regenerated by the action of arylsulfatase (ARS) enzymes (7). The ARS enzyme family currently comprises six members (ARSA to ARSF) (10), and a major ARS known as steroid sulfatase or ARSC is present in the endoplasmic reticulum of many tissues (11, 12, 13), including the placenta, where its main function is in estrogen biosynthesis (14). ARSC has been demonstrated to hydrolyze iodothyronine sulfates, whereas ARSA and ARSB do not (15) (Visser, T. J., et al., manuscript in preparation). The expression of particular SULT and ARS enzymes in individual cells and tissues can therefore provide a sensitive and responsive means of controlling the activity of potent endogenous chemicals in target and nontarget cells.
Despite the important role of sulfation, little is known of the detailed ontogeny of SULT and ARS enzymes in humans. We have previously examined the ontogeny of the major steroid SULT enzyme [dehydroepiandrosterone (DHEA) sulfotransferase, SULT2A1] in liver, adrenal, and kidney (16) and showed that in liver the expression of the enzyme increases during development, whereas in the adrenal gland the expression is high (>5-fold higher than in liver) from at least 12 weeks gestation, consistent with the critical role of the fetal adrenal in the production of DHEA sulfate to serve as substrate for placental estrogen biosynthesis. Using immunohistochemistry and semiquantitative immuno-dot blot analysis, we have also showed that in the developing lung, phenol (family 1) SULT expression is high early in gestation and reduces after birth, whereas steroid SULT (SULT2A1) expression is low in early fetal life but increases with development to reach adult levels near term (17). Thus the expression of various SULT isoforms appears to be differentially regulated during human development.
Of the many endogenous chemicals for which sulfation is a key metabolic event, thyroid hormone and catecholamines are among the most important, since these compounds dramatically influence human development. Sulfation of iodothyronines has profound effects on their metabolism by iodothyronine deiodinases and on their homeostasis (18, 19). Once sulfated, T4 is exclusively metabolized to the inactive rT3 and cannot be converted to the receptor-active T3 (18). Sulfation also accelerates the degradation (deiodination) of T3 and the major metabolic product of iodothyronine metabolism 3,3'-diiodothyronine (3, 3'-T2) is also extensively sulfated (18). Thus sulfation is likely to play a key role in regulating the amount of receptor-active thyroid hormone in target tissues. Iodothyronine sulfates are present at very high levels in fetal (cord) blood and persist well into postnatal life, supporting the importance of sulfation as a means of protecting the fetus from excessive T3 ( (20, 21); our unpublished observations). Similarly, catecholamines are important mediators of the body’s response to physiological stresses such as hypoxia and hypoglycemia, and catecholamine secretion is believed to play an important role in immediate adaptation to extra-uterine life, including metabolic, respiratory and cardiovascular events (22). Animal studies, principally in sheep, indicate that stresses such as hypoxia and birth produce dramatic increases in fetal catecholamine production (23). Indeed, the dramatic surge in catecholamine secretion at birth may be a key event since catecholamine infusion to the fetal sheep mimics the immediate postnatal profiles of glucagon and insulin and consequently gluconeogenesis (22). In adult humans, the vast majority of catecholamines such as dopamine, noradrenaline and adrenaline are present in the circulation in the form of their respective sulfate conjugates (24) - e.g. plasma concentrations of free dopamine (<0.1 pmol/mL) represent less than 1% of the total combined free and sulfate-conjugated dopamine. Sulfate conjugates may be thought of as biologically inactive forms of these biogenic amines, which provide a source of the active, free compounds in target tissues through enzymatic hydrolysis as well as providing an appropriate route of excretion.
To further our understanding of the role of sulfation of catecholamines and thyroid hormone during human development, we have studied the ontogeny of the SULT and ARS isoenzymes involved in their metabolism in key tissues, liver, lung and brain using isoform-selective probe substrates and immunochemical techniques. Our data strongly support the idea that the human fetus has an extensive capacity for sulfation, and we demonstrate for the first time a developmentally programmed switch in the hepatic expression of the major catecholamine-metabolizing sulfotransferase (SULT1A3).
Materials and Methods
Materials
All chemicals were of analytical grade and obtained from either Sigma, Poole, UK or Merck, Glasgow, UK. [35S]3'-Phosphoadenosine 5'-phosphosulfate (PAP35S) (1.57 Ci/mmol), [2,4,6,7-3H (N)]estrone (70 Ci/mmol) and [1,2,6,7-3H(N)]dehydroepiandrosterone (83 Ci/mmol) were purchased from Du Pont/NEN Life Science Products, Stevenage, UK 3, (3'-125I)T2 was prepared by radioiodination of 3-T1 as previously described (25). 3,3'-T2S and 3, (3'-125I) T2S were prepared as described by Mol and Visser (26).
Tissues
Ethical approval was obtained from the Ethics Committee of Tayside Health Board and subsequently the Tayside Committee on Medical Research Ethics. Tissue (liver, lung, brain) was obtained from fetuses (10–22 weeks gestation, without evidence of structural abnormality) within 6 h following termination of pregnancy using Gemeprost vaginal pessaries. Infant tissue (26–85 weeks post conception) was obtained at routine postmortem within 12 h of certification of death. Tissues were either snap frozen in liquid nitrogen and stored at -80 C or used immediately for the preparation of microsomes and cytosols. To assess the postmortem stability of SULT enzyme activity in liver samples, livers from 3 fetuses (1 at 13 weeks gestation and 2 at 14 weeks gestation) were removed within 30 min and a portion of each liver (approx. 20% of total) immediately snap frozen in liquid nitrogen and stored at -80 C - this was taken to represent a time of 0 h postmortem for subsequent analysis. The remainder of each liver was wrapped in 2 12-ply 10 cm x 10 cm swabs moistened with sterile 0.9% (wt/vol) saline, placed in a large sterile Petri dish and stored at 4 C. At 3-h intervals, up to a total of 12 h postmortem, an additional 20% of each liver was removed, frozen in liquid nitrogen, and stored at -80 C. Cytosolic fractions were prepared, and SULT enzyme activity toward 4-nitrophenol and dopamine was determined as described below.
Preparation of microsomes and cytosols and enzyme assays
Tissues were homogenized in 20% (wt/vol) 0.25 mol/L sucrose, 10 mmol/L HEPES (pH 7.4), and 3 mmol/L 2-mercaptoethanol, and microsomes and cytosol were harvested by differential centrifugation as previously described (27). Microsomes and cytosol were either used immediately or stored at -80 C until used (within 3 months).
It is possible to identify individual SULT isoforms in tissue cytosols using specific substrates: SULT1A1 (4-nitrophenol), SULT1A3 (dopamine), SULT1E1 (estrone), and SULT2A1 (DHEA). Sulfation of estrone (7.1 µmol/L), DHEA (2.7 µmol/L), and 3,3'-T2 (0.1 µmol/L) by cytosolic sulfotransferases was determined using radioactive substrates as described previously (28, 29, 30). Sulfation of 4-nitrophenol (3.3 µmol/L) and dopamine (4.7 µmol/L) was determined using 0.4 µmol/L PAP35S (0.08 µCi) as originally described by Foldes and Meek (31), and assay conditions were optimized with respect to pH, cytosolic protein content, and incubation time. Sulfatase activity was measured in microsomal preparations using 0.1 µmol/L (0.05 µCi) [125I]T2 sulfate ([125I]T2S) as substrate. Reactions (37 C) were started by the addition of enzyme in ice-cold buffer (0.1 mol/L Tris-Cl-, pH 7.0) and were stopped by the addition of 0.8 mL 0.1 mol/L HCl. The mixtures were analyzed for 3,3'-T2 formation by chromatography on Sephadex LH-20 minicolumns as previously described (32). Desulfation in complete reaction mixtures was corrected for background radioactivity detected in the corresponding Sephadex LH-20 fractions of control incubations without enzyme. The protein content of microsomal and cytosolic preparations was estimated by the method of Bradford (33), using BSA as standard.
SDS-PAGE and immunoblot analysis
Cytosol fractions were resolved on 11% (acrylamide monomer) SDS-polyacrylamide gels and transferred to nitrocellulose as first described by Laemmli (34) and Towbin et al. (35), respectively. Antibodies against purified, recombinant human SULT1A3 (36) were raised in a sheep by three separate immunizations (4 weeks apart) with 150 µg protein (Scottish Antibody Production Unit, Carluke, Scotland). Antiserum was shown to react with recombinant human SULT1A3 and SULT1A1 by enzyme-linked immunosorbent assay and Western immunoblot analysis, and the IgG fraction of the antiserum was purified by chromatography on a column of protein G-agarose (Pierce Chemical Co., Chester, UK). Immunochemical detection of SULT1A1 and SULT1A3 proteins on immunoblots of human tissue cytosols was achieved using the enhanced chemiluminescence method as described by the manufacturer (Amersham Pharmacia Biotech, Aylesbury, UK).
Immunohistochemistry
Formalin-fixed, paraffin-embedded tissue was cut in sequential 3-µm sections. The first section was stained with hematoxylin and eosin. Immunohistochemistry was performed on the second section using rabbit anti-(rat SULT1A1) IgG (37) at a final concentration of 1.25 mg/ml. This antibody preparation has been shown to recognize members of the human SULT1 family in liver by immunoblot analysis (37, 38) and in the developing human kidney by immunohistochemistry (39). A final section was used as a negative control with replacement of the primary antibody by nonimmune IgG. Human fetal kidney sections (16–19 weeks gestation) were used as positive controls, as the anti-(rat SULT1A1) antibody preparations have previously been shown to have discrete and intense immunoreactivity in this renal tissue (39). A standard peroxidase-antiperoxidase technique was used incorporating 3,3'-diaminobenzidine as a developing agent (40). Sections were lightly counterstained with hematoxylin, dehydrated through graded alcohols, and cleared in xylene before coverslipping in synthetic resin.
Results
Postmortem stability of SULT enzyme activities in human fetal liver
A potential source of variability in enzyme activity in such
studies is the quality of the tissue and the postmortem interval. To
determine whether the postmortem interval significantly influenced SULT
enzyme activity, we designed an experiment in which freshly isolated
fetal liver was stored at 4 C, and samples were taken for preparation
of cytosolic fraction and SULT enzyme assay over a 12-h period. The
results (Fig. 1
) show that SULT1A1 and
SULT1A3 activities were stable over the subsequent 12-h postmortem
interval, with approximately 80% of enzyme activity remaining after
this time. The majority of this modest reduction, which was not
statistically significant, occurred in the period between 9 and 12
h postmortem. Similarly, a pilot study (Visser, T. J.,
unpublished) performed a number of years ago to measure
T2 SULT activity in human fetal liver showed no
association between postmortem interval and enzyme activity up to
20 h postmortem. It is unlikely, therefore, that the variation in
enzyme activity measured in the population results from differences in
postmortem interval-related enzyme stability.
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SULT activity was determined toward the substrates dopamine,
4-nitrophenol and 3,3'-T2, in cytosols from fetal
and neonatal livers and lungs (Table 1).
Fetal liver displayed substantial SULT activity toward all substrates
examined, and these activities were generally reduced in the postnatal
liver cytosols. The presence of significant SULT1A3 activity (with
dopamine as substrate) in the fetal liver was surprising, because this
activity is essentially absent from adult human liver
(41). Sulfation of dopamine in the postnatal liver was
reduced to less than 10% of fetal values, suggesting a programmed
developmental switch in expression of the SULT1A3 enzyme. The fetal
lung also demonstrated significant SULT1A1 (4-nitrophenol) and SULT1A3
(dopamine) activities. Iodothyronine sulfation (3,
3'-T2 as preferred substrate) was significantly
lower in fetal lung than in fetal liver. In postnatal lung cytosols,
all activities were reduced compared with fetal values. Detailed
ontogenic profiles of SULT activity toward dopamine, 4-nitrophenol, and
3,3'-T2 are shown in Fig. 2.
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). There was no correlation
between the sulfation of dopamine and that of
3,3'-T2 by fetal and postnatal liver cytosols
(Fig. 3A); however, there was a positive correlation
(r2 = 0.39; P < 0.0001, by
linear regression analysis) between 3,3'-T2 and
4-nitrophenol sulfation (Fig. 3B), suggesting that the SULT1A1 enzyme
is a major enzyme responsible for the sulfation of this iodothyronine
during human development. When only the postnatal samples
(i.e. where SULT1A3 is not expressed) were included in the
analysis, the correlation was much greater (r2 =
0.78; P < 0.0001).
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Cytosols were prepared from tissue dissected from eight major
brain structures and were assayed for iodothyronine sulfation (with
3,3'-T2) and for sulfation of the isoform-
selective SULT substrates 4-nitrophenol and dopamine (Table 2) to determine the expression pattern of
these SULT1A isoforms in fetal brain. The most striking feature was the
substantial sulfation of 4-nitrophenol and
3,3'-T2 in the choroid plexus of the lateral
ventricle, whereas dopamine sulfation was not detectable at this
location. Sulfation of dopamine was low throughout the fetal brain,
with activity highest in the germinal eminence and cerebellum. There
was a significant correlation (r2 = 0.90) between
the sulfation of 4-nitrophenol and that of
3,3'-T2 in the different brain regions, again
supporting a primary role for SULT1A1 in the sulfation of
3,3-T2.
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Human SULT1A1 and -1A3 share more than 93% amino acid sequence
identity, and the antibodies we prepared against recombinant SULT1A3
cross-reacted with SULT1A1 on enzyme-linked immunosorbent assay (not
shown) and immunoblot analysis. It was possible to use this antibody
preparation to confirm the expression of these two enzymes in
developing liver and brain (Fig. 4), as
the two isoforms have different electrophoretic mobilities on SDS-PAGE.
In the liver (Fig. 4A), SULT1A1 is expressed at equivalent levels in
fetal and postnatal periods and at an increased level in adult liver,
consistent with enzyme activity measurements [the average adult liver
SULT1A1 activity measured with 4-nitrophenol as substrate in the
presence of 0.4 µmol/L PAPS was approximately 130 pmol/min·mg in
our hands (Gilissen, R. A. H. J., and M. W. H.
Coughtrie, unpublished observations)]. In contrast, SULT1A3 expression
was high in the fetus, dramatically reduced in postnatal liver, and
essentially absent from adult liver cytosol.
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), a very
strong band corresponding to SULT1A1 expression was detected in the
choroid plexus, consistent with the extensive sulfation of
4-nitrophenol and 3,3'-T2. Faint bands
corresponding to SULT1A1 and SULT1A3 were detectable in cytosols from
most regions of the fetal brain, consistent with the overall low levels
of SULT activity measured (Table 2). 3,3'-T2 sulfatase activity in developing human tissues
Iodothyronine sulfatase activity (with
3,3'-T2 sulfate) was measured in microsomal
preparations from fetal and postnatal liver and lung (Table 3). Activities were low in both tissues,
and hepatic activities were considerably higher than those in lung.
There was a trend toward decreased activity in postnatal compared with
fetal samples. We also prepared microsomes from different fetal brain
regions and determined 3,3'-T2S sulfatase
activity (Table 4). Activities were very
low in each region, and there was no evidence of selective expression
in any particular area.
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Using anti-(rat SULT1A1) IgG we examined the expression of SULT1A
family members in human fetal liver (16 weeks gestation) by
immunohistochemistry (Fig. 5). At this
stage of human development, the liver comprises both hepatocytes and
hemopoietic cells, and immunoreactivity was present in both cell types,
with a granular distribution through the cytoplasm. The intensity of
staining in the hepatocytes was considerably weaker than that in the
hemopoietic cells (Fig. 5A), which are easily identifiable by their
small, dense nuclei (Fig. 5, B and C). In contrast, adult liver is
devoid of these hemopoietic cells, and staining is therefore confined
to hepatocytes. This suggests that a significant portion of the enzyme
activity measured in cytosols prepared from human fetal liver
(e.g. Table 1) results from SULT1A enzyme(s) present in
these hemopoietic cells.
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The developing human is dependent upon numerous powerful hormonal signals for the regulation of key phases of tissue differentiation and maturation. However, it is also necessary to have mechanisms for protecting target and nontarget cells and tissues from overstimulation by these chemical mediators, and sulfation appears to have evolved to play an important role in this process in humans. The human fetus expresses various SULT enzymes from very early in gestation; SULT2A1 is expressed in hepatocytes at 32 postovulatory days (16), and SULT1A enzyme(s) is detectable in mesonephric kidney at 32 postovulatory days (39) and in lung epithelium at 56 postovulatory days (17). However, the expressions of these various enzymes are under specific temporal and spatial regulation, which indicates that these (and presumably other) tissues have different requirements for terminating or modulating these signaling chemicals during development.
Here we conducted a detailed analysis of the expression of two major SULT isoforms involved in the metabolism of iodothyronines and catecholamines in developing human liver, brain, and lung and also quantified arylsulfatase activity in these tissues with 3,3'-T2S as substrate. These studies reveal novel and important features of the sulfation system in human development. Most interestingly, the catecholamine-metabolizing SULT enzyme SULT1A3 is expressed at high levels in human fetal liver, in contrast to the adult liver, where it is virtually absent. We found extremely reduced levels of enzyme activity and protein expression in postnatal liver samples, suggesting that in the perinatal period, SULT1A3 expression is actively (and probably rapidly) turned off. More detailed analysis of the site of expression of SULT1A isoforms using immunohistochemistry showed that although there was expression in fetal hepatocytes, a significant portion of the immunoreactivity was concentrated in the hemopoietic cells, which make up a substantial part of the fetal liver during the second trimester. On about the 35th day of gestation, erythropoiesis begins in the liver and between the 12th and 20th weeks, and erythroid precursors represent approximately 50% of the total nucleated cells of this organ (42). As the antibodies used for immunohistochemistry cross-react with both SULT1A1 and SULT1A3 (and also presumably SULT1A2, as it shares >95% amino acid sequence identity), it is not possible to tell from these studies which isoform(s) contributes to the immunoreactivity in the hepatic hemopoietic cells. Immunoblot analysis and enzyme assays showed that SULT1A1 expression was similar in fetal and postnatal liver cytosols, whereas SULT1A3 expression was substantially reduced in the postnatal liver compared with the fetal tissue, and it is possible therefore that SULT1A3 expression predominates in the hemopoietic cells. We have previously shown that these hemopoietic cells do not express the major steroid sulfotransferase, SULT2A1 (16). It is also possible that this antibody cross-reacts with another SULT isoform that is expressed at high levels in fetal hemopoetic cells. The physiological relevance of this high level of SULT1A3 expression in fetal liver is unclear. However catecholamines, which are major endogenous substrates for this enzyme, play a critical role in immediate adaptation to the extrauterine environment (22), and at birth there are large increases in catecholamine production, which is believed to modulate blood pressure and the glucose homeostasis necessary for the transition to postnatal life. We believe it is reasonable to propose that the expression of SULT1A3 in fetal liver has a protective function against the biological activity of catecholamines, and that because the neonate must be able to fully respond to catecholamines, it is necessary to remove this protection before birth through switching off SULT1A3 expression. It is, of course, possible that SULT1A3 has an as yet unknown function in the hemopoietic cells of the fetal liver and that the reduced expression with advancing gestation parallels the disappearance of these cells from the liver.
The SULT1A1 enzyme was not subject to the same developmental regulation
in liver as SULT1A3 [or, for that matter, SULT2A1, which increased
during fetal development and continued to do so postnatally
(16)]. Hepatic SULT1A1 enzyme activity was present from
10 weeks gestation (the earliest samples studied), but was extremely
variable over the developmental range. One possible source of this
variation is a common polymorphism in the SULT1A1 gene,
resulting in an Arg213
His mutation in the
protein (43), which appears to affect enzyme activity and
expression levels in human platelets and probably other tissues
(44, 45). Another possible source of variation is
differences in the quality of tissue, resulting from the range of
postmortem intervals encountered. We attempted to assess the
contribution of this phenomenon by sampling freshly obtained liver over
a period of 12 h postmortem and determining SULT1A1 and SULT1A3
enzyme activities. The results clearly show that the enzyme activities
are stable at least up to 12 h postmortem, suggesting that
instability of enzyme protein resulting from variable postmortem
intervals is unlikely to be a major factor in the observed variability
of enzyme activity. The variability most likely arises from a
combination of genetic and environmental influences on enzyme activity
and expression. Expression levels did not vary significantly between
fetal and postnatal liver, although by adulthood levels are
approximately 2-fold higher. Comparison of the sulfation of 4-
nitrophenol, dopamine, and 3,3'-T2 clearly
demonstrated a significant relationship between 4-nitrophenol and
3,3'-T2 sulfation, suggesting that the SULT1A1
enzyme is principally responsible for the sulfation of this
iodothyronine in human liver. This assumption is supported by data from
in vitro kinetic experiments with recombinant human SULTs
(46). The fetal lung also expressed SULT1A3 and, to a
lesser extent, SULT1A1, and these enzyme activities were substantially
reduced in the postnatal samples. These results are consistent with our
previous immunochemical studies, which showed that SULT1A family enzyme
protein expression decreased with advancing developmental age in the
human lung (17).
The expression of these enzymes from early in human gestation again suggests that sulfation is important for the developing fetus. Both SULT1A1 and SULT1A3 have very broad substrate specificities, including iodothyronines (predominantly 3,3'-T2 and T3) and many xenobiotics, thus they may have an important detoxification function during the critical stages of development. This is particularly important in the absence of the other major detoxifying enzyme system, UDP-glucuronosyltransferase (3). Numerous chemical procarcinogens and promutagens (including food-derived heterocyclic amines) are bioactivated by SULT1A1, and SULT1A3 is required for the activation of certain dietary compounds, such as estragole (47). We have previously determined that human fetal and neonatal liver cytosol is able to sulfate hydroxyaryl amines and aryl hydroxamic acids (48). These enzymes may therefore be a potential source of DNA- and protein-adducting agents after maternal exposure to such compounds.
Investigation of the expression of SULT1A1 and SULT1A3 in the developing human brain yielded interesting information. SULT1A3 is expressed at low levels in the fetal brain, although the germinal eminence appeared to be the principal site of expression; this is where the majority of neuroblast cell division occurs in the developing mammalian brain. The SULT1A1 enzyme was also expressed at low levels in most brain regions, but again the choroid plexus was a major site of expression. Selective expression of this enzyme in the choroid plexus of the fetal brain is intriguing. The choroid plexus is of neuroectodermal origin, as is the rest of the brain, and its primary function is the production of cerebrospinal fluid. It is also the most highly vascularized tissue of the developing brain, and therefore, as a potential portal of entry of circulating toxins that may result from maternal exposure, it seems reasonable for this tissue to have a high degree of chemical defense. Other enzymes of detoxification (UDP-glucuronosyltransferases, cytochromes P450) have been demonstrated in the choroid plexus and blood-brain interfaces in adult rats (49, 50).
We have also quantified, for the first time, arylsulfatase activity toward a sulfated iodothyronine (3,3'-T2S in this case) during human development. Hydrolysis of sulfate conjugates is an important component of the overall sulfation system, as it provides the facility to regenerate the biologically active parent compound in a target tissue. The concentrations of sulfated iodothyronines (T3S, T4S, and rT3S) are extremely high in the human fetal circulation (51) (Hume, R., T. J. Visser, M. W. H. Coughtrie, and F. L. R. Williams, unpublished), much higher than in the adult, although the exact source of these conjugates remains to be determined. There is thus a potentially large pool of thyroid hormone that would be dependent on the hydrolytic activity of sulfatase enzyme(s). Both 3,3'-T2S and T3S appear to be substrates for the microsomal ARSC, or steroid sulfatase, enzyme (Kester, M. H. A., E. Kaptein, M. W. H. Coughtrie, and T. J. Visser, in preparation), which is expressed at high levels in the placenta (14, 52). We observed that fetal liver, and to a much lesser extent fetal lung and brain, expressed this iodothyronine sulfatase activity and thus have the potential to generate free iodothyronines from their respective sulfate conjugates.
Sulfation is obviously important during human development and probably performs multiple functions in protection of the fetus against the toxic effects of endogenous and xenobiotic chemicals. The data presented here extend our studies on the developmental regulation of SULT expression during human development and confirm that in some tissues certain enzymes are tightly regulated, indicating specific temporal requirements for sulfation. Further investigations are required to more clearly delineate the roles of reversible sulfation during human development and to identify the factors responsible for the differential regulation of expression of these important enzymes in humans.
Acknowledgments
We are grateful to Dr. Graeme Eisenhofer, NIH, for helpful discussions.
Footnotes
1 This work was supported by grants from the Wellcome Trust (to
R.H.), Tenovus Scotland (to M.W.H.C. and R.H.), and the Chief
Scientist’s Office (to R.H. and M.W.H.C.) and by an equipment grant
from the Wellcome Trust (to M.W.H.C.). 
2 Present address: Quantase Ltd., 3 Riverview Business Park, Perth,
Scotland, United Kingdom PH2 8DE.
Received January 26, 2000.
Revised July 5, 2000.
Revised November 30, 2000.
Accepted February 27, 2001.
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 F. L. R. Williams, G. J. Mires, C. Barnett, S. A. Ogston, H. van Toor, T. J. Visser, R. Hume, and with collaboration from the Scottish Preterm Thyro Transient Hypothyroxinemia in Preterm Infants: The Role of Cord Sera Thyroid Hormone Levels Adjusted for Prenatal and Intrapartum Factors J. Clin. Endocrinol. Metab., August 1, 2005; 90(8): 4599 - 4606. [Abstract] [Full Text] [PDF]
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 V. M. Arlt, M. Stiborova, C. J. Henderson, M. R. Osborne, C. A. Bieler, E. Frei, V. Martinek, B. Sopko, C. R. Wolf, H. H. Schmeiser, et al. Environmental Pollutant and Potent Mutagen 3-Nitrobenzanthrone Forms DNA Adducts after Reduction by NAD(P)H:Quinone Oxidoreductase and Conjugation by Acetyltransferases and Sulfotransferases in Human Hepatic Cytosols Cancer Res., April 1, 2005; 65(7): 2644 - 2652. [Abstract] [Full Text] [PDF]
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 N. Hempel, H. Wang, E. L. LeCluyse, M. E. McManus, and M. Negishi The Human Sulfotransferase SULT1A1 Gene Is Regulated in a Synergistic Manner by Sp1 and GA Binding Protein Mol. Pharmacol., December 1, 2004; 66(6): 1690 - 1701. [Abstract] [Full Text] [PDF]
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R. Hume, J. Simpson, C. Delahunty, H. van Toor, S. Y. Wu, F. L. R. Williams, and T. J. Visser Human Fetal and Cord Serum Thyroid Hormones: Developmental Trends and Interrelationships J. Clin. Endocrinol. Metab., August 1, 2004; 89(8): 4097 - 4103. [Abstract] [Full Text] [PDF]
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N. Murphy, R. Hume, H. van Toor, T. G. Matthews, S. A. Ogston, S.-Y. Wu, T. J. Visser, and F. L. R. Williams The Hypothalamic-Pituitary-Thyroid Axis in Preterm Infants; Changes in the First 24 Hours of Postnatal Life J. Clin. Endocrinol. Metab., June 1, 2004; 89(6): 2824 - 2831. [Abstract] [Full Text] [PDF]
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M. Stiborova, M. Miksanova, S. Smrcek, C. A. Bieler, A. Breuer, K. A. Klokow, H. H. Schmeiser, and E. Frei Identification of a genotoxic mechanism for 2-nitroanisole carcinogenicity and of its carcinogenic potential for humans Carcinogenesis, May 1, 2004; 25(5): 833 - 840. [Abstract] [Full Text] [PDF]
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H. Glatt and W. Meinl Use of genetically manipulated Salmonella typhimurium strains to evaluate the role of sulfotransferases and acetyltransferases in nitrofen mutagenicity Carcinogenesis, May 1, 2004; 25(5): 779 - 786. [Abstract] [Full Text] [PDF]
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 M. H. A. Kester, E. Kaptein, T. J. Roest, C. H. van Dijk, D. Tibboel, W. Meinl, H. Glatt, M. W. H. Coughtrie, and T. J. Visser Characterization of rat iodothyronine sulfotransferases Am J Physiol Endocrinol Metab, September 1, 2003; 285(3): E592 - E598. [Abstract] [Full Text] [PDF]
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V. M. Arlt, H. Glatt, E. Muckel, U. Pabel, B. L. Sorg, H. H. Schmeiser, and D. H. Phillips Metabolic activation of the environmental contaminant 3-nitrobenzanthrone by human acetyltransferases and sulfotransferase Carcinogenesis, November 1, 2002; 23(11): 1937 - 1945. [Abstract] [Full Text] [PDF]
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 C. A. Strott Sulfonation and Molecular Action Endocr. Rev., October 1, 2002; 23(5): 703 - 732. [Abstract] [Full Text] [PDF]
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 M. H. A. Kester, E. Kaptein, C. H. Van Dijk, T. J. Roest, D. Tibboel, M. W. H. Coughtrie, and T. J. Visser Characterization of Iodothyronine Sulfatase Activities in Human and Rat Liver and Placenta Endocrinology, March 1, 2002; 143(3): 814 - 819. [Abstract] [Full Text] [PDF]
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 C. R. Ireson, D. J. L. Jones, S. Orr, M. W. H. Coughtrie, D. J. Boocock, M. L. Williams, P. B. Farmer, W. P. Steward, and A. J. Gescher Metabolism of the Cancer Chemopreventive Agent Curcumin in Human and Rat Intestine Cancer Epidemiol. Biomarkers Prev., January 1, 2002; 11(1): 105 - 111. [Abstract] [Full Text]
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E. L. Stanley, R. Hume, T. J. Visser, and M. W. H. Coughtrie Differential Expression of Sulfotransferase Enzymes Involved in Thyroid Hormone Metabolism during Human Placental Development J. Clin. Endocrinol. Metab., December 1, 2001; 86(12): 5944 - 5955. [Abstract] [Full Text] [PDF]
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) and dopamine (SULT1A3; 
) was
measured in cytosolic fractions prepared from fetal liver samples
stored at 4 C for up to 12 h, as described in Materials and
Methods. Data points represent the mean ±
SD for enzyme activity determinations performed in
triplicate on three separate liver samples. Mean enzyme activity values
(±SD) at time zero were 172 ± 39 pmol/min·mg for
SULT1A1 and 73 ± 6 pmol/min·mg for SULT1A3. No statistically
significant differences between enzyme activity at 3, 6, 9, or 12
h postmortem and activity at 0 h were observed, as assessed by
Student’s t test (two-tailed; Prism software,
GraphPad, Inc., San Diego, CA).
