Structure-activity relationships for hydroxylated polychlorinated biphenyls as inhibitors of the sulfation of dehydroepiandrosterone catalyzed by human hydroxysteroid sulfotransferase SULT2A1

Article abstract of DOI:10.1021/tx200260h

Polychlorinated biphenyls (PCBs) are persistent worldwide pollutants that are of concern due to their bioaccumulation and health effects. Metabolic oxidation of PCBs results in the formation of hydroxylated metabolites (OHPCBs). Among their biological effects, OHPCBs have been shown to alter the metabolism of endocrine hormones, including inhibition of mammalian cytosolic sulfotransferases (SULTs) that are responsible for the inactivation of thyroid hormones and phenolic steroids (i.e., hSULT1A1, hSULT1B1, and hSULT1E1). OHPCBs also interact with a human hydroxysteroid sulfotransferase that plays a role in the sulfation of endogenous alcohol-containing steroid hormones and bile acids (i.e., hSULT2A1). The objectives of our current study were to examine the effects of a series of OHPCB congeners on the activity of hSULT2A1 and to develop a three-dimensional quantitative structure-activity relationship (3D-QSAR) model for OHPCBs as inhibitors of the enzyme. A total of 15 OHPCBs were examined, and the sulfation of 1 μM [³H] dehydroepiandrosterone (DHEA) was utilized as a model reaction catalyzed by the enzyme. All 15 OHPCBs inhibited the sulfation of DHEA, with IC₅₀ values ranging from 0.6 μM to 96 μM, and eight of these OHPCBs were also substrates for the enzyme. Comparative molecular field analysis (CoMFA) provided a predictive 3D-QSAR model with a q² value of 0.697 and an r ² value of 0.949. The OHPCBs that had the highest potency as inhibitors of DHEA sulfation were those with a 3, 5-dichloro-4-hydroxy substitution pattern on the biphenyl ring system, and these congeners were also substrates for sulfation catalyzed by hSULT2A1.

Full text of DOI:10.1021/tx200260h

ARTICLE
pubs.acs.org/crt
StructureꢀActivity Relationships for Hydroxylated Polychlorinated
Biphenyls As Inhibitors of the Sulfation of Dehydroepiandrosterone
Catalyzed by Human Hydroxysteroid Sulfotransferase SULT2A1
Edugie J. Ekuase,† Yungang Liu,† Hans-Joachim Lehmler,‡ Larry W. Robertson,‡ and Michael W. Duffel*,†
†Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, and ‡Department of Occupational and
Environmental Health, College of Public Health, The University of Iowa, Iowa City, Iowa 52242, United States
S Supporting Information
b
ABSTRACT: Polychlorinated biphenyls (PCBs) are persistent
worldwide pollutants that are of concern due to their bioaccu-
mulation and health effects. Metabolic oxidation of PCBs results
in the formation of hydroxylated metabolites (OHPCBs).
Among their biological effects, OHPCBs have been shown to
alter the metabolism of endocrine hormones, including inhibi-
tion of mammalian cytosolic sulfotransferases (SULTs) that
are responsible for the inactivation of thyroid hormones and
phenolic steroids (i.e., hSULT1A1, hSULT1B1, and hSULT1E1).
OHPCBs also interact with a human hydroxysteroid sulfotransferase that plays a role in the sulfation of endogenous alcohol-
containing steroid hormones and bile acids (i.e., hSULT2A1). The objectives of our current study were to examine the effects of a
series of OHPCB congeners on the activity of hSULT2A1 and to develop a three-dimensional quantitative structureꢀactivity
relationship (3D-QSAR) model for OHPCBs as inhibitors of the enzyme. A total of 15 OHPCBs were examined, and the sulfation of
1 μM [3H] dehydroepiandrosterone (DHEA) was utilized as a model reaction catalyzed by the enzyme. All 15 OHPCBs inhibited
the sulfation of DHEA, with IC50 values ranging from 0.6 μM to 96 μM, and eight of these OHPCBs were also substrates for the
enzyme. Comparative molecular field analysis (CoMFA) provided a predictive 3D-QSAR model with a q2 value of 0.697 and an r2
value of 0.949. The OHPCBs that had the highest potency as inhibitors of DHEA sulfation were those with a 3, 5-dichloro-4-hydroxy
substitution pattern on the biphenyl ring system, and these congeners were also substrates for sulfation catalyzed by hSULT2A1.
’ INTRODUCTION
andsulfationmightbeexpectedtoleadtoeliminationoftheOHPCB,
the actual fates of these conjugates are less clear since some PCB
sulfates and glucuronides retain significant lipophilic properties
based on the calculated octanol/water partition coefficients.14
The concentrations of OHPCBs have been determined in
humans,15ꢀ20 and there is evidence for selective retention of
OHPCBs in blood relative to the parent PCBs.15 These hydro-
xylated metabolites have been implicated in some of the adverse
health effects observed upon exposure to PCBs. For example,
some OHPCBs bind with high affinity to the thyroid hormone
transport protein transthyretin,21 and this may contribute to
metabolic disruptions in thyroid hormone function in some
tissues. The OHPCBs are also potent inhibitors of several
cytosolic sulfotransferases (SULTs) that are important in the
metabolism of endocrine hormones. These SULTs include the
family 1 enzymes hSULT1A1 and hSULT1B1 that catalyze
sulfation of thyroid hormones.22,23 Inhibition of another family
1 isoform, the estrogen sulfotransferase (hSULT1E1), by OHPCBs
can lead to increased levels of estrogens in tissues, thus pro-
viding a mechanism for the estrogenic effects seen with these
molecules.24
Polychlorinated biphenyls (PCBs) are synthetic chemicals
that were produced and sold for decades. Although production
of PCBs was banned in the late 1970s, these compounds are a
persistent worldwide problem.1ꢀ5 Many of the PCBs with higher
numbers of chlorine atoms (e.g., 5 or more) are relatively re-
sistant to biodegradation, accumulate in the food chain, and have
significant health effects in humans and other animals.6 Those
PCBs with lower numbers of chlorine atoms can be released into
the atmosphere from various sources that include, among others,
contaminated buildings, water, and soil.1 Significant levels of
volatile PCBs have been measured in both indoor and outdoor
air.7ꢀ12 These airborne PCBs are inhaled, and there is evidence
of increased blood concentrations of the lower chlorinated PCBs
in humans.5 Metabolism of these lower chlorinated PCBs can
lead to reactive intermediates that are implicated in carcinogen-
esis and other toxicities.1,5,13
Mammalian metabolism of PCBs often involves oxidation
catalyzed by cytochrome P450 (CYP) enzymes to form hydro-
xylated PCBs (OHPCBs).6 Further metabolism of OHPCBs
may include conjugation reactions such as those catalyzed by
cytosolic sulfotransferases (SULTs) and UDP-glucuronosyltran-
sferases (UGTs), with the resulting formation of PCB sulfates
and PCB glucuronides, respectively.6,14 While glucuronidation
Received: June 24, 2011
Published: September 13, 2011
r
2011 American Chemical Society
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Figure 1. Structures of OHPCBs used in this study. The numbering scheme for 40-OH PCB 3 is indicated.
The interactions of OHPCBs with family 2 SULTs, such as
hSULT2A1 (previously known as the human hydroxysteroid, or
alcohol, sulfotransferase) are, however, not as well understood.
The hSULT2A1 plays an important role in the sulfation of
alcohol-containing steroids, bile acids, and xenobiotics.25ꢀ27
Previous studies in our laboratory showed that three OHPCBs
interact with hSULT2A1,28 although structureꢀactivity relation-
ships were not available from that work. Therefore, we have now
more extensively examined the relationships between the struc-
tures of OHPCBs and their interactions with hSULT2A1 as
inhibitors and substrates.
BL21 (DE3) cells using the following minor modification of a previously
described procedure.31 After sterile Luria broth (LB) medium was sup-
plemented with filter-sterilized ampicillin to a final concentration of
50 μg/mL, a 3 mL volume of this medium was inoculated with cells and
incubated at 28 °C for 24 h on a reciprocating shaker at 210 rpm.
Aliquots of 100 μL each from this culture were added to each of four
culture tubes containing 20 mL of LB medium. Following incubation
for 24 h under the above conditions, the contents of each tube were
added to four 1 L flasks that each contained 400 mL of sterile LB
medium, and the cells were incubated for 1 h at 28 °C with shaking at
210 rpm. Cells were then induced by adding 1 mM IPTG (isopropyl-
1-thio-D-galactopyranoside) and incubated under the above condi-
tions for 23 h. The resulting cells were harvested by centrifugation at
10,000g for 30 min. The cell pellet (19 g, wet weight) was suspended
in 20 mL of ice-cold buffer containing 0.25 M sucrose, 10% (v/v)
glycerol, 1 mM phenylmethyl sulfonyl fluoride, 1 μM pepstatin A,
1 mM dithiothreitol, 3.3 μM antipain, and 10 mM Tris-HCl at pH 7.5.
The cells were disrupted by sonication with a Branson Digital Sonifier
(model 450, Branson Ultrasonics Corp., Danbury, CT) and then sub-
jected to centrifugation for 30 min at 12,000g. The resulting supernatant
fraction was stored at ꢀ70 °C until purification. Purification of hSULT2A1
was accomplished using the previously described method.32 The pur-
ified enzyme was homogeneous as judged by SDSꢀPAGE with staining
by Coomassie Blue. Protein concentrations were determined by the
modified Lowry procedure33 with bovine serum albumin as the standard.
The specific activity of hSULT2A1 during purification steps was deter-
mined using the methylene blue paired ion extraction assay.34,35
’ MATERIALS AND METHODS
Chemicals and Reagents. All OHPCBs (structures shown in
Figure 1) were synthesized as described previously.29 30-Phosphoade-
nosine-50-phosphosulfate (PAPS) was purchased from Sigma-Aldrich
(St. Louis, MO), and it was further purified (>98% as analyzed by
HPLC) using a previously published procedure.30 3H-DHEA (94.5 Ci/
mmol) was obtained from Perkin-Elmer Life and Analytical Sciences
(Boston, MA). Econo-Safe biodegradable scintillation cocktail and Tris-
HCl were purchased from RPI (Mt. Prospect, IL). Hydroxyapatite (Bio-
Gel HT) was from Bio-Rad Laboratories (Hercules, CA), and Tween 20
was obtained from J.T. Baker Chemicals (Phillipsburg, NJ). All other
chemicals were of the highest purity commercially available.
Expression and Purification of Human SULT2A1. Human
sulfotransferase hSULT2A1 was expressed in recombinant Escherichia coli
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Inhibition of hSULT2A1 by OHPCBs. The ability of OHPCBs to
inhibit the sulfation of DHEA catalyzed by hSULT2A1 was determined
using a previously described radiochemical assay method.36 Assays
contained 200 μM PAPS, 1 μM [3H]DHEA (final radioactive specific
activity of 2 μCi/nmol), varying concentrations of OHPCBs in ethanol
(final ethanol concentration of less than 2%, v/v), 0.25 M potassium
phosphate at pH 7.0, and 7.5 mM 2-mercaptoethanol in a total volume of
200 μL. After the addition of 0.25 μg of hSULT2A1 to start the reaction,
the assay mixture was incubated for 10 min at 37 °C. The reaction was
terminated by the addition of 0.8 mL of 50 mM KOH, unreacted DHEA
was extracted with 0.5 mL of CHCl3, and radioactivity of the DHEA-
sulfate in a 100 μL of aliquot of the aqueous layer was determined. The
rate of sulfation was expressed as nmoles of DHEA sulfate formed per
minute per mg of protein. The means of three determinations at each
concentration of inhibitor were fit to a sigmoidal doseꢀresponse curve
(SigmaPlot, v.11; Systat Software, Chicago, IL) to determine IC50 values.
OHPCBs As Substrates for hSULT2A1. A previously described
HPLC assay35,37 was used to determine the OHPCBs that were substrates
for hSULT2A1. Reaction mixtures (total volume of 30 μL) contained
200 μM PAPS, 0.25 M potassium phosphate at pH 7.0, 7.5 mM
2-mercaptoethanol, and OHPCBs dissolved in acetone (final concen-
tration of acetone less than 5%, v/v). Sulfation reactions were initiated
by the addition of 1 μg of enzyme and incubated at 37 °C for 10 min.
Reactions were terminated by the addition of 30 μL of methanol followed
by votex-mixing for 30 s. The resulting reaction mixture was kept on ice
until analysis of the substrate-dependent formation of the product,
adenosine 30,50-diphosphate (PAP), was carried out by HPLC. The limit
of detection for this assay method is 0.1 nmol of PAP formed/min/mg of
purified recombinant hSULT2A1.31
3D-QSAR Model for the Inhibition of hSULT2A1 by
OHPCBs. Comparative molecular field analysis (CoMFA)38 was used
to generate predictive models for 15 OHPCBs. Structures of these
OHPCBs were constructed in Sybyl 8.0. (Tripos Inc., 2008) and saved in
MOL2 format. GasteigerꢀHuckel charges were assigned to the com-
pounds, and the gradient convergence criterion set at 0.05 kcal/(Åmol).
Alignment was done on a common biphenyl substructure with 4-OHPCB
68 as the template. The IC50 values were converted into ꢀlog values and
served as the dependent variables in the CoMFA analysis. After alignment,
each molecule was analyzed in a three-dimensional cubic lattice with an
sp3 carbon atom probe having a net charge of +1 and energy cutoff value
of 30 kcal/mol. The CoMFA steric field, calculated according to the
LennardꢀJones potential, and the electrostatic field, calculated by the
Coulomb potential, were obtained. CoMFA models were derived, and
they were evaluated using leave-one-out (LOO) cross-validation. Values
for q2 (predictive power of the model), the optimum number of com-
ponents, and r2 (correlation coefficient for fit of the data set activity to
the model) were then obtained. External validation of the method was
carried out by the removal of 3 of the compounds and generation of a
second model based on the remaining 12 compounds.
Table 1. Inhibition of Human SULT2A1 by OHPCBsa
compound
IC50 (μM)
4-OHPCB 34
40-OHPCB 68
40-OHPCB 33
40-OHPCB 25
4-OHPCB 36
40-OHPCB 9
40-OHPCB 6
4-OHPCB 8
0.6 ( 1.2
0.8 ( 1.3
2.5 ( 1.1
4.4 ( 1.3
4.4 ( 1.2
7.5 ( 1.1
9.8 ( 1.1
10 ( 1.2
13 ( 1.1
14 ( 1.2
16 ( 1.1
16 ( 1.1
16 ( 1.1
28 ( 1.1
96 ( 1.2
4-OHPCB 14
4-OHPCB 11
40-OHPCB 12
60-OHPCB 35
30,40-diOHPCB 5
40-OHPCB 3
30,40-diOHPCB 3
a The structures of these compounds are shown in Figure 1. IC50 values
are the micromolar concentration resulting in 50% inhibition of the
sulfation of DHEA catalyzed by hSULT2A1. IC50 values are expressed as
the means and standard error of the fit of the data in Figure S1
(Supporting Information) based on triplicate determinations of the rate
of sulfation at each concentration of inhibitor.
atom ortho to the ring junction (i.e., 40-OHPCB 9, 40-OHPCB 6,
and 4-OHPCB 8) were generally good inhibitors, with IC50
values of 8ꢀ10 μM. Furthermore, the dichlorinated OHPCBs
with both chlorine atoms in the nonphenolic ring (i.e., 40-OHPCB
9 and 40-OHPCB 12) indicated that the position of the ortho
chlorine atom at the ring junction in 40-OHPCB 9 increased the
potency of inhibition of hSULT2A1 when compared with that of
the non-ortho analogue, 40-OHPCB 12.
The overall patterns indicating both the importance of an ortho
chlorine at the ring junction and the favorable presence of a 3,5-
dichloro-4-hydroxy substitution pattern for inhibition were also
observed with the trisubstituted OHPCBs. For example, com-
parison of 4-OHPCB 34 with 4-OHPCB 36 indicated an ap-
proximately 7-fold decrease in IC50 upon changing the chlorine
atom in the nonphenolic ring from the meta to ortho position
with respect to the ring junction. While the 30,40-diOHPCBs
were among the least potent inhibitors, the presence of a chlorine
atom ortho to the ring junction in 30,40-diOHPCB 5 did have an
effect consistent with the observed differences in IC50 values for
other OHPCBs with and without ortho chlorine atoms.
Sulfation of OHPCBs Catalyzed by hSULT2A1. Since alter-
nate substrates for an enzyme might be responsible for the inhibition
of a specific reaction such as the sulfation of DHEA catalyzed by
hSULT2A1, we investigated the possibility that some of these
OHPCBs might be substrates for sulfation catalyzed by the enzyme.
Indeed, 8 of the 15 OHPCBs were substrates for hSULT2A1.
The initial velocity data for these compounds were fitted to a
substrate inhibition equation v = Vmax/(1+ (Km/[S]) + [S]/Kis)
(Table 2). A concentration of 200 μM PAPS was used due to
previous findings that the highest initial velocities at low con-
centrations of DHEA were achieved with 100ꢀ300 μM PAPS.36
The values for Km (i.e., the concentration of substrate at half
maximal velocity) ranged from 8.1 ( 3.2 μM to 56.8 ( 58.2 μM
with 4-OHPCB 34 having the lowest Km value. The values for
Vmax (i.e., the maximum reaction velocity) ranged from 8.5 (
0.5 nmol/min/mg to 57.1 ( 11.4 nmol/min/mg. These data
’ RESULTS
OHPCBs As Inhibitors of the Sulfation of DHEA Catalyzed
by hSULT2A1. As seen in Table 1, all of the OHPCBs studied
(structures in Figure 1) inhibited the hSULT2A1-catalyzed
sulfation of DHEA, and there was a greater than 100-fold range
in IC50 values. Complete dose-dependent inhibition curves for
each OHPCB are provided in the Supporting Information
(Figure S1). Among the 15 OHPCBs examined, the two most
potent inhibitors of the sulfation of DHEA were OHPCB 34 and
40-OHPCB 68, with IC50 values of 0.6 and 0.8 μM, respectively.
These two compounds possess a 3,5-dichloro-4-hydroxy sub-
stitution pattern, and they also have a chlorine atom ortho to the
biphenyl ring junction. Dichlorinated OHPCBs with a chlorine
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Table 2. Kinetic Parameters for the Sulfation of OHPCBs Catalyzed by hSULT2A1
OHPCB
Vmax (nmol/min/mg)
Km (μM)
Kis (μM)
kcat/Km (minꢀ1mMꢀ1
)
4-OHPCB 14
4-OHPCB 34
4-OHPCB 36
4-OHPCB 8
40-OHPCB 33
40-OHPCB 25a
4-OHPCB 11
44.3 ( 3.9
57.1 ( 11.4
51.8 ( 16.3
56.1 ( 42.6
21.0 ( 7.4
8.5 ( 0.5
8.5 ( 1.8
220 ( 56
47.4 ( 16.2
39.4 ( 23.1
65.4 ( 73.8
217 ( 189
-
176
238
129
33
8.1 ( 3.2
13.6 ( 6.7
56.8 ( 58.2
26.2 ( 16.2
8.6 ( 2.0
27
33
21.4 ( 18.3
36.6 ( 43.9
51.9 ( 65.7
20
a While 40-OHPCB 25 and 40-OHPCB 68 (not shown) were substrates for hSULT2A1, reliable kinetic constants for Kis (both OHPCBs) and for Km and
V
max (40-OHPCB 68) were not obtained due to low solubility. Values of Km and Vmax for 40-OHPCB 25 were estimated using the MichaelisꢀMenten
equation. Note: kcat/Km values are based on a subunit Mr of 33,785.
Figure 2. Ability of purified recombinant hSULT2A1 to catalyze sulfation of OHPCBs. Assays were carried out as described in Materials and Methods.
(A) Sulfation of 4-OHPCB 14 ((), 4-OHPCB 34 ()), 4-OHPCB 8 (b), 40-OHPCB 33 (0), and 4-OHPCB 11 (1). (B, C, and D) Sulfation of
40OHPCB 68, 4-OHPCB 36, and 40-OHPCB 25, respectively. Data are fit to the following equation for substrate inhibition: v = Vmax/(1 + (Km/[S]) +
[S]/Kis). Data points are the means ( standard error of triplicate determinations.
indicated that hSULT2A1 had approximately 10 times higher
catalytic efficiency (kcat/Km) for OHPCBs with a 3,5-dichloro-4-
hydroxy substitution pattern, i.e., 4-OHPCB 14, 4-OHPCB 34, and
4-OHPCB 36. As observed in the kinetic profiles shown in Figure 2,
at higher concentrations of substrate, a decrease in rate is seen due to
substrate inhibition. The values calculated for the substrate inhibi-
tion constant, Kis, provide a quantitative measure of this effect. As it
has been shown previously for DHEA, this substrate inhibition in
hSULT2A1 is likely the result of the formation of dead-end
complexes (e.g., an enzymeꢀPAPꢀsubstrate complex).36 There
are, however, differences in the magnitude of kinetic values for
OHPCBs as substrates for hSULT2A1 when compared with those
for DHEA at the same 200 μM concentration of PAPS. Kinetic
values for Vmax, Km, and Kis with DHEA as substrate were 245 (
40 nmol/min/mg, 0.8 ( 0.3 μM, and 8 ( 3 μM, respectively.36
Development of 3D-QSAR Models for the Interaction of
OHPCBs with hSULT2A1. Comparative molecular field analysis
(CoMFA) was employed to develop a three-dimensional quan-
titative structureꢀactivity relationship for the OHPCB-mediated
inhibition of the sulfation of DHEA catalyzed by hSULT2A1.
The IC50 values that were obtained from the inhibition studies
(Table 1) were converted into pIC50 (ꢀlog IC50 (M)) values for
the CoMFA analysis. The biphenyl core structure of each OHPCB
was aligned with a template molecule (40-OHPCB 68) in order to
get the conformation for correlation analysis and model devel-
opment. The template molecule, 40-OHPCB 68, was chosen
following comparison to other OHPCBs that might serve as
template. This template had the lowest number of components
with a reasonable q2 value, and parameters following the com-
parison of other potential template structures are provided in the
Supporting Information (Table S1). The superimposition of all
the compounds on the template molecule, 40- OHPCB 68 is
shown in Figure 3. The CoMFA model derived using this template
had a steric contribution of 38% and an electrostatic contribution
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Figure 3. Superimposition of all 15 hydroxylated polychlorinated biphenyls (OHPCBs) with 40-OHPCB 68 as the template molecule using a common
substructure-based alignment.
Table 3. Experimental IC50 Values Compared to Predicted
IC50 Values in the CoMFA of 15 OHPCBs (Model 1)
IC50
actual
pIC50
predicted
pIC50
compd
(μM)
differencea
40-OHPCB 3
40-OHPCB 6
4-OHPCB 8
28.1
9.8
4.551
5.009
5.000
5.125
4.854
4.801
4.886
5.357
5.602
6.222
4.810
5.357
6.097
4.019
4.799
4.525
5.117
5.303
5.114
4.843
4.763
5.082
5.273
5.503
6.022
4.721
5.268
6.153
3.947
4.853
0.026
0.108
0.303
0.011
0.011
0.039
0.196
0.084
0.099
0.199
0.088
0.088
0.056
0.072
0.054
10.0
7.5
40-OHPCB 9
4-OHPCB 11
40-OHPCB 12
4-OHPCB 14
40-OHPCB 25
40-OHPCB 33
4-OHPCB 34
60-OHPCB 35
4-OHPCB 36
40-OHPCB 68
30,40-diOH PCB 3
30,40-diOH PCB 5
14.0
15.8
13.0
4.4
2.5
Figure 4. Actual versus predicted values of ꢀlog IC50 for the set of 15
OHPCBs as inhibitors of the sulfation of DHEA catalyzed by hSUL-
T2A1. Circled data points indicate those OHPCBs that also serve as
substrates for sulfation catalyzed by hSULT2A1.
0.6
15.5
4.4
0.8
95.7
15.9
a Represents the difference between the experimental and the predicted
values for pIC50 (i.e., ꢀlog IC50).
of 62% with q2 of 0.697, r2 of 0.949, and a standard error (SE) of
0.159. The differences between the actual pIC50 values and the
predicted pIC50 values were less than 0.5 (Table 3). As seen in
Figure 4, there was a good correlation observed between the
experimental and predicted values for pIC50. Also noted in Figure 4,
the most potent inhibitors of DHEA sulfation catalyzed by
hSULT2A1 were also substrates for the enzyme.
Although the leave-one-out cross-validation parameter q2
provides a measure of the predictive power of the 3D-QSAR
model, it is also useful to analyze the ability of the procedure to
predict IC50 values with more than one compound that is
not among the compounds used for developing the CoMFA
model. Thus, we removed 4-OHPCB 14, 4-OHPCB 25, and
4-OHPCB 12 from the training set and analyzed an additional
CoMFA model developed with the remaining 12 compounds
(Figure 5). The IC50 values predicted from this model for
4-OHPCB 12 and 4-OHPCB 25 were similar to the experi-
mental IC50 values obtained (Table 4), and there was a 3.8-fold
Figure 5. Actual versus predicted values of ꢀlog IC50 for the set of 12
OHPCBs (Model 2).
difference between the predicted and the actual IC50 values for
4-OHPCB 14. The model had a q2 of 0.540, r2 of 0.957, and a
SE of 0.163 at 4 components. The steric contribution and
the electrostatic contributions were 29% and 71%, respectively.
Therefore, the external validation process confirmed that the
overall methodology employed for CoMFA model development
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Table 4. Predicted and Experimental Values of IC50 for 3
OHPCBs Using a CoMFA for 12 OHPCBs (Model 2)a
the sulfation of a substrate (e.g., DHEA) catalyzed by the
hSULT2A1.
The finding that the best inhibitors of the enzyme were also
alternative substrates and that there is a seamless correlation in
IC50 values between those OHPCBs that were alternate sub-
strates and those that were solely inhibitors of the enzyme is
indicative of a similarity in interactions occurring with the enzyme.
Since substrates must bind at the active site for the sulfation
reaction to occur, it is reasonable to assume that all of the
OHPCBs examined bind at the active site of the enzyme. This
should not, however, be interpreted to imply that the mode of
inhibition is competitive since there are numerous examples
where binding at the active site of an enzyme can display a non-
competitive mode of inhibition.44 Indeed, difficulties in directly
comparing our IC50 data with kinetic constants for sulfation of
the OHPCBs that are substrates imply a more complex behavior
than would be expected for simple competitive inhibition. Such
complex behavior is common with homodimeric bisubstrate
enzymes, where the interaction of a substrate/inhibitor might
occur with multiple forms of the enzyme that result either from
the formation of different ternary complexes or from conforma-
tional changes in the enzyme subsequent to the binding of one
substrate molecule. Binding of the first substrate molecule can then
affect binding of a second substrate molecule either to a second
active site in the homodimer or to an allosteric site. Indeed, dif-
ferences in ternary enzyme complexes and conformational changes
in hSULT2A1 upon binding of DHEA to the homodimer have been
reported.36,45ꢀ47 Thus, an OHPCB substrate/inhibitor binding to
the sulfurylacceptor siteinhSULT2A1mightinduce similar confor-
mational changes with kinetic constants that would deviate from
those expected for simple competitive inhibition. While we cannot
yet verify or rule out such interactions with the OHPCBs, additional
studies to more fully determine the nature of these binding
interactions of OHPCBs with hSULT2A1 are in progress. More-
over, differences in the formation of dead-end enzyme complexes36
as well as conformational changes36,45,46 have also been implicated
in substrate inhibition observed with DHEA. As recently seen in
comparisons of DHEA and raloxifene,46 there are mechanistic
differences in hSULT2A1-catalysis that are dependent upon the
structure of the substrate. Thus, interactions with OHPCBs may
also involve structure-dependent differences that lead to alterations
in modes of binding and substrate inhibition, and further inves-
tigation will be required to elucidate these relationships.
Consideration of the in vivo significance of the sulfation of
OHPCBs in humans is currently limited to in vitro studies inter-
preted in the context of serum concentrations of OHPCBs, the
presence of specific SULTs in relevant tissues, the substrate specifi-
cities of these enzymes, and the concentrations of endogenous
substrates for the SULTs. As an example of endogenous substrate
for hSULT2A1, DHEA is present at a human serum concentra-
tion that varies with several factors, but it is usually within a range
of 5ꢀ24 nM.48 Androsterone, another substrate for hSULT2A1,
has been reported to be present at 2.5ꢀ5.0 nM in serum.48
Concentrations of OHPCBs in blood have often been reported
on the basis of serum or plasma fresh weights, while tissue
concentrations of OHPCBs have often been based on lipid weight.
This causes difficulties in making direct comparisons. However,
we have used previously reported concentrations of 0.082ꢀ0.328
ng/g49 and 0.117ꢀ11.6 ng/g20 based on whole blood wet weight
in humans to estimate an approximate range of OHPCB concen-
trations of 0.3ꢀ40 nM.28 Comparison of these concentrations with
Km values for DHEA indicate that both DHEA and OHPCBs are
predicted
predicted
actual
test compds
(pIC50
)
actual (pIC50)
IC50 (μM) IC50 (μM)
40-OHPCB 12
4-OHPCB 25
4-OHPCB 14
4.73
5.39
5.47
4.80
5.36
4.89
18.6
4.10
3.37
15.8
4.40
13.0
a Three compounds were removed from the training set in Model 1, and
a second, related, CoMFA model was derived based on the 12 remaining
compounds. Model 2 was then used to predict IC50 values for 40-
OHPCB 12, 4-OHPCB 25, and 4-OHPCB 14.
was predictive for the inhibition of sulfation of DHEA catalyzed
by hSULT2A1.
’ DISCUSSION
The cytosolic sulfotransferases catalyze sulfation reactions
that are involved in the regulation of hormones, detoxication
of bile acids and xenobiotics, activation of therapeutic drugs to
obtain desired pharmacological effects, and conversion of xeno-
biotics to more reactive products that can cause cancer and other
toxic responses.39ꢀ43 As detailed in the Introduction, the poten-
tial roles of sulfation in endocrine disruption, carcinogenesis, and
other toxicities of the hydroxylated metabolites of PCBs are of
increasing interest. We are particularly interested in the interac-
tion of the sulfotransferases with those OHPCB metabolites
containing lower numbers (e.g., fewer than five) of chlorine
atoms since these may be formed metabolically following ex-
posure to the parent PCBs in contaminated indoor and outdoor
air. The studies presented here were specifically focused on the
interactions of OHPCBs with hSULT2A1 due to the importance
of this family 2 SULT in the metabolism of alcohol-containing
steroid hormones and bile acids.
Structural characteristics of OHPCBs, due to the number and
substitution pattern of the chlorine atoms, are important deter-
minants of their overall metabolism and toxicity. Since the large
number of potential OHPCB congeners precludes a detailed
examination of each of the lower chlorinated OHPCBs, it is
important to develop models that will be useful in predicting the
effect of OHPCBs on hSULT2A1 based on fundamental struc-
tural characteristics. Such models will be particularly useful in
assessing the potential for individual OHPCB congeners to
interfere with the sulfation of hormones and other endogenous
substrates for hSULT2A1 as well as determining the potential for
interference with the metabolism of drugs and other xenobiotics.
Preliminary attempts to correlate IC50 values with either the
acidꢀbase constants of the OHPCBs or their octanol/water
partition coefficients did not provide predictive models (data not
shown). As a result, we turned to CoMFA in order to develop a
3D-QSAR model for the interaction of OHPCBs with hSUL-
T2A1. A set of 15 OHPCBs was used to determine their ability to
inhibit the catalysis of sulfation of DHEA by hSULT2A1. Those
OHPCBs with 3,5-dichloro-4-hydroxy substitutions proved to be
the most potent inhibitors, and they were also substrates for
sulfation reactions catalyzed by hSULT2A1. Additionally, the
presence of a chlorine atom ortho to the ring junction in the
OHPCB enhanced the inhibition of sulfation of DHEA. Whether
the OHPCB served as an alternate substrate or solely as an inhibitor,
the CoMFA model provided a mechanism for the prediction
of the ability of a lower chlorinated OHPCB to interfere with
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Chemical Research in Toxicology
ARTICLE
present at concentrations less than their Km values (i.e., the rate
of the hSULT2A1-catalyzed reaction is directly dependent upon
the concentration of OHPCB or DHEA). Thus, our observation
that some OHPCBs inhibit the sulfation of DHEA at concentra-
tions lower than the nonsaturating concentration of DHEA used
in our assays implies that they may compete effectively for DHEA
as substrate for hSULT2A1 in some cells. These results point to a
need for future examination of the potential in vivo importance
through the use of intact human cell systems as well as develop-
ment of methods for the extraction and analysis of the sulfated
metabolites from human blood.
Although our present study focused on the inhibition of the
sulfation of DHEA, these interactions and the predictive capacity
of CoMFA models would likely hold for other steroid substrates
for hSULT2A1 (e.g., pregnenolone and androsterone). This would
be particularly important when assessing either the potential for
endocrine disruption by this mechanism or alteration in the
metabolism of drugs or other xenobiotics through the inhibition
of hSULT2A1. Additionally, we would expect from our findings
that those OHPCBs that are the best substrates for hSULT2A1
would be predicted to be the best inhibitors of the enzyme in its
catalysis of the sulfation of DHEA, other steroid substrates, or
other xenobiotics. While the model(s) derived in this study
proved to be useful in the prediction of the ability of OHPCBs to
inhibit the sulfation of DHEA catalyzed by hSULT2A1, there are
some potential limitations that should be considered. The models
did not include highly chlorinated OHPCBs, particularly those
that have two or more chlorines ortho to the ring junction.
Furthermore, the highly chlorinated OHPCBs may have addi-
tional structural interactions with hSULT2A1 that are different
from those of the OHPCBs included in the present work. Although
an attempt was made to examine one such higher chlorinated
OHPCB (i.e., 4-OHPCB 165) in the context of these studies, this
was not possible due to the limited solubility of this compound
under the assay conditions.
Funding Sources
This study was supported by the National Institutes of Health
through research grants P42 ES013661 (to L.W.R., M.W.D., H.-J.L.)
from the National Institute of Environmental Health Sciences
and R01 CA38683 (to M.W.D.) from the National Cancer
Institute. We also acknowledge programmatic support through
the University of Iowa Environmental Health Sciences Research
Center (NIEHS/NIH P30 ES05605).
’ DISCLOSURE
The contents of this paper are solely the responsibility of the
authors and do not necessarily represent the official views of
the NIH.
’ ABBREVIATIONS
CoMFA, comparative molecular field analysis; DHEA, dehy-
droepiandrosterone; DHEAS, dehydroepiandrosterone sulfate;
DE52, diethylaminoethyl cellulose anion exchanger; DTT,
dithiothreitol; HPLC, high performance liquid chromatography;
hSULT2A1, human hydroxysteroid sulfotransferase 2A1; IPTG,
isopropyl-1-thio-D-galactopyranoside; LB, Luria broth; OHPCB,
hydroxylated polychlorinated biphenyl; 40-OHPCB 3, 40-hydro-
xy-4-monochlorobiphenyl; 40-OHPCB 6, 40-hydroxy-2,30-di-
chlorobiphenyl; 4-OHPCB 8, 4-hydroxy-2,40-dichlorobiphenyl;
40-OHPCB 9, 40-hydroxy-2,5-dichlorobiphenyl; 4-OHPCB 11,
4-hydroxy-3,30-dichlorobiphenyl;40-OHPCB 12, 40-hydroxy-3,4-
dichlorobiphenyl; 4-OHPCB 14, 4-hydroxy-3,5-dichlorobiphe-
nyl; 40-OHPCB 25, 40-hydroxy-2,30,4-trichlorobiphenyl; 40-OHPCB
33, 40-hydroxy-20,3,4-trichlorobiphenyl; 4-OHPCB 34, 4-hydro-
xy-20,3,5-trichlorobiphenyl; 60-OHPCB 35, 60-hydroxy-3,30-4 tri-
chlorobiphenyl; 4-OHPCB 36, 4-hydroxy-3,30,5-trichlorobiphenyl;
40-OHPCB 68, 40-hydroxy-2,30,4,50-tetrachlorobiphenyl; 30,
40-diOHPCB 3, 30,40-dihydroxy-4-monochlorobiphenyl; 30,40-
diOHPCB 5, 30,40-dihydroxy-2,3-dichlorobiphenyl; PAP, adeno-
sine 30,50-diphosphate; PAPS, 30-phosphoadenosine 50-phospho-
sulfate; PCB, polychlorinated biphenyl; SULT, cytosolic sulfo-
transferase; 3D-QSAR, three-dimensional quantitative structureꢀ
activity relationship; Tris-HCl, [tris(hydroxymethyl)aminomethane]
hydrochloride.
In summary, we have examined the ability of OHPCBs to
inhibit the sulfation of DHEA catalyzed by hSULT2A1 and deter-
mined those cases where the inhibition was due to the OHPCB
serving as an alternate substrate for hSULT2A1. We have also
developed a 3D-QSAR model that will aid in predicting the
actions of other similar OHPCBs on hSULT2A1. Such models
will be important in determining the role of lower chlorinated
OHPCBs in the disruption of sulfation of endogenous and xeno-
biotic molecules. As more information is learned about the biolo-
gical activities/toxicities of the PCB-sulfates, these models may
also assist in predicting which lower chlorinated PCB congeners
would potentially pose a threat to human health through mechan-
isms involving the formation of PCB-sulfates.
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