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Research Article
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In Vitro Inhibition of Human Hepatic and cDNA-expressed Sulfotransferase Activity with 3-Hydroxybenzo[a]pyrene by Polychlorobiphenylols Li-Quan Wang,1 Hans-Joachim Lehmler,2 Larry W.
Robertson,2 Charles N. Falany,3 and Margaret O. James1 1Department of Medicinal Chemistry, University of Florida, Gainesville,
Florida, USA; 2Department of Occupational and Environmental Health,
College of Public Health, University of Iowa, Iowa City, Iowa, USA; 3Department
of Pharmacology and Toxicology, University of Alabama, Birmingham, Alabama,
USA Abstract Sulfonation is a major phase II biotransformation reaction. In this study, we found that several polychlorobiphenylols (OH-PCBs) inhibited the sulfonation of 3-hydroxybenzo[a]pyrene (3-OH-BaP) by human liver cytosol and some cDNA-expressed sulfotransferases. At concentrations > 0.15 µM, 3-OH-BaP inhibited its own sulfonation in cytosol fractions that were genotyped for SULT1A1 variants, as well as with expressed SULT1A1*1, SULT1A1*2, and SULT1E1, but not with SULT1A3 or SULT1B1. The inhibition fit a two-substrate kinetic model. We examined the effects of OH-PCBs on the sulfonation of 0.1 or 1.0 µM 3-OH-BaP, noninhibitory and inhibitory substrate concentrations, respectively. At the lower 3-OH-BaP concentration, OH-PCBs with a 3-chloro-4-hydroxy substitution pattern were more potent inhibitors of cytosolic sulfotransferase activity [with concentrations that produced 50% inhibition (IC50) between 0.33 and 1.1 µM] than were OH-PCBs with a 3,5-dichloro-4-hydroxy substitution pattern, which had IC50 values from 1.3 to 6.7 µM. We found similar results with expressed SULT1A1*1 and SULT1A1*2. The OH-PCBs were considerably less potent inhibitors when assay tubes contained 1.0 µM 3-OH-BaP. The inhibition mechanism was noncompetitive, and our results suggested that the OH-PCBs competed with 3-OH-BaP at an inhibitory site on the enzyme. The OH-PCBs tested inhibited sulfonation of 3-OH-BaP by SULT1E1, but the order of inhibitory potency was different than for SULT1A1. SULT1E1 inhibitory potency correlated with the dihedral angle of the OH-PCBs. The OH-PCBs tested were generally poor inhibitors of SULT1A3- and SULT1B1-dependent activity with 3-OH-BaP. These findings demonstrate an interaction between potentially toxic hydroxylated metabolites of PCBs and polycyclic aromatic hydrocarbons, which could result in reduced clearance by sulfonation. Key words: 3-hydroxy-benzo[a]pyrene, human liver cytosol, inhibition of sulfonation, polychlorobiphenylols, SULT1A1*1, SULT1A1*2, SULT1E1. Environ Health Perspect 113:680-687 (2005) . doi:10.1289/ehp.7837 available via http://dx.doi.org/ [Online 24 February 2005] Address correspondence to M.O. James, Department of Medicinal Chemistry, Room P6-20B, 1600 SW Archer Rd., University of Florida, Gainesville, FL 32610-0485 USA. Telephone: (352) 846-1952. Fax: (352) 846-1972. E-mail: mojames@ufl.edu We thank F.P. Guengerich for providing samples of human liver and W. Farmerie for providing access to instruments for polymerase chain reaction amplification. This study was supported by grants P42 ES07375 and P42 ES07380 from the National Institute of Environmental Health Sciences, National Institutes of Health (NIH) , and grant GM38954 from the National Institute of General Medical Sciences, NIH. This article reflects the authors’ views and not any official views of NIH. The authors declare they have no competing financial interests. Received 8 December 2004 ; accepted 24 February 2005. |
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Polycyclic aromatic hydrocarbons (PAHs) and polychlorinated
biphenyls (PCBs) are two classes of environmentally prevalent
pollutants. PAHs are formed through the combustion of fossil
fuels and the burning of organic materials (Dipple 1985). PCBs
were first produced industrially in the middle of the last century
for their desirable dielectric properties (Erickson 2001) and
remain in the environment because of their continued use, because
of their release from waste sites, and because many congeners
are slowly degraded. The more lipophilic PAHs and PCBs are often
found in the same environmental samples, such as soils and sediments,
and are biotransformed in animals by similar pathways (James
2001).
Of the PAHs, benzo[a]pyrene (BaP) is a well-studied
chemical carcinogen, which is metabolized by cytochrome P-450
(CYP) to a variety of products (Dipple 1985). These include 3-hydroxybenzo[a]pyrene
(3-OH-BaP), a major metabolite of BaP in humans and animals,
which has estrogenic properties and binds to hemoglobin (Charles
et al. 2000; Sugihara and James 2003). Hydroxylated PAH metabolites
such as 3-OH-BaP are substrates for glucuronidation and sulfonation,
catalyzed by one or more of the UDP-glucuronosyltransferases
and 3´-phosphoadenosine 5´-phosphosulfate (PAPS)-dependent
sulfotransferases (SULTs), respectively (James et al. 2001).
Sulfonation is considered a detoxification pathway for 3-OH-BaP.
PCBs have several metabolites of toxicologic importance, including
the polychlorobiphenylols (OH-PCBs), which are formed in vivo from
CYP-dependent mono-oxygenation of PCBs (James 2001). Although
they are slightly more hydrophilic than are the parent PCBs,
several OH-PCBs are eliminated slowly (Klasson-Wehler et al.
1993). People who are highly exposed to PCBs through the diet
typically have OH-PCBs in their blood, some bound to plasma proteins
(Guvenius et al. 2003; Sandau et al. 2000). Several OH-PCB congeners
interact with components of the endocrine system, potentially
interfering with thyroid hormone and estrogen function (Lans
et al. 1993; Safe 1994; Schuur et al. 1998). Although the OH-PCBs
have low affinities for both and β estrogen
receptors, some OH-PCBs are strikingly potent inhibitors of human
estrogen sulfotransferase (SULT1E1), with subnanomolar concentrations
that produced 50% inhibition (IC50) (Kester et al.
2000). This suggests that OH-PCBs may be indirectly estrogenic
by increasing estradiol bioavailability in target tissues. As
well as possibly causing toxicity by inhibiting the sulfonation
of hormones, several OH-PCBs inhibited the sulfonation and glucuronidation
of the PAH metabolite 3-OH-BaP in channel catfish intestine (van
den Hurk et al. 2002).
Sulfonation is an important phase II conjugation pathway for
the detoxification of xenobiotics as well as the modulation of
endogenous compounds such as thyroid hormones, steroids, and
neurotransmitters (Coughtrie et al. 1998). One or more members
of a superfamily of cytosolic SULT enzymes catalyze these reactions
(Blanchard et al. 2004). SULT1A1, SULT1B1, and SULT1E1 are the
major phenol sulfotransferases expressed in human liver, with
SULT1A1 (also known as ST1A3) found at the highest concentration
(Honma et al. 2002). SULT1A3 is expressed in the gut but is present
in very low concentrations in adult human liver (Richard et al.
2001). Genetic polymorphisms are known for SULT1A: a G638 A
transition leading to an Arg213 His
exchange in the protein was observed with a frequency of 33.2%
in Caucasian subjects, 8% in Chinese, and 29.4% in African Americans
(Carlini et al. 2001). SULT1A1*His (SULT1A1*2) was a less thermostable
protein than SULT1A1*Arg (SULT1A1*1), and some authors have reported
that the SULT1A1*2 variant is less catalytically active (Ozawa
et al. 1998; Raftogianis et al. 1997).
Because people are frequently coexposed to PAHs and PCBs, we
wished to determine if OH-PCBs would inhibit 3-OH-BaP sulfonation
in human liver (HL) cytosol and, if so, whether the inhibition
was isozyme selective. We used cDNA-expressed human SULT1A1*1,
-1A1*2, -1A3, -1B1, and 1E1 isozymes, which we expected would
use 3-OH-BaP as substrate. We genotyped the HL cytosol fractions
used in this study, with respect to the common SULT1A1 polymorphic
variants, to examine the possibility that OH-PCBs would affect
their activity differently. These studies were conducted with
a series of predominantly para-OH-PCBs.
![Figure 1](fig1sm.gif)
Figure 1. Structures of the hydroxylated PCBs
used in this study. Type A, hydroxy without a flanking
chlorine atom; type B, para-hydroxy with one flanking
chlorine atom; type C, para-hydroxy with two flanking
chlorine atoms.
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Materials. The structures of the OH-PCBs used
in this study are shown in Figure 1. In naming these OH-PCBs,
we followed the recommendation of Maervoet et al. (2004) to
name them as metabolites of PCBs, referring back to the Ballschmiter
and Zell numbering system for PCBs (Ballschmiter and Zell 1980).
The 6´-OH-CB35 (A1), 4´-OH-CB35 (B1), 4´-OH-CB36
(B2), 4´-OH-CB79 (C1), and 4-OH-CB36 (C2) were synthesized
by Suzuki coupling as described previously (Bauer et al. 1995;
Lehmler and Robertson 2001). We verified the structures of
each of these OH-PCBs by 1H and 13C-nuclear
magnetic resonance spectroscopy, Fourier transform infrared
spectroscopy, and gas chromatography-mass spectrometry (GC-MS).
We found that
each OH-PCB was > 99% pure by GC-MS analysis (Mass Spectrometry
Facility, University of Kentucky, Lexington, KY), combustion
analysis (Atlantic Microlab, Atlanta, GA), and thin-layer chromatography.
The 4´-OH-CB69 (B3), 4´-OH-CB106 (B4), 4´-OH-CB112
(B5), 4´-OH-CB121 (C3), 4´-OH-CB159 (C4), 4´-OH-CB165
(C5), and 4´-OH-CB72 (C6) were purchased from AccuStandard
(New Haven, CT). S.S. Singer (University of Dayton, Dayton,
OH) supplied the PAPS. We purchased 35S-PAPS, 3.05 µCi/nmol
(99.1% pure), from PerkinElmer Life Science (Boston, MA). Benzo[ a]pyrene-3-sulfate
(BaP-3-SO 4) and 3-hydroxybenzo[ a]pyrene (3-OH-BaP)
were purchased from the NCI Chemical Carcinogen Reference Standard
Repository (Midwest Research Institute, Kansas City, MO). We
obtained HaeII from Fisher Scientific (Atlanta, GA) and Taq DNA
polymerase, along with other polymerase chain reaction (PCR)
reagents, from Promega (Madison, WI). Integrated DNA Technologies
(Coralville, IA) supplied primers for use in genotyping. We
purchased the highest available grade of other reagents from
Fisher Scientific
(Atlanta, GA) and Sigma Chemical Company (St. Louis, MO).
Physicochemical properties of the OH-PCBs. We
calculated the structural characteristics of dihedral angle,
molecular volume, molecular surface area, pKa,
log P, and log D at pH 7.0 with MM2* using GB/SA
water solvent continuum as implemented by MacroModel 5.0 (Schrödinger,
Portland, OR) and described previously by Tampal et al. (2002).
Cytosolic preparations. F.P. Guengerich (Vanderbilt
University) kindly donated the samples of human liver, which
were procured from organ donors (Guengerich 1995). We prepared
liver cytosolic fractions from four livers by standard methods
and stored aliquots at -80°C
until use (Wang et al. 2004). We used three or four of these
cytosol fractions in each experiment.
SULT1A1 genotype determination. We used
a genomic DNA isolation kit (EASY-DNA; InVitrogen, Carlsbad,
CA) to extract genomic DNA from samples of the individual human
livers used in this study. We used a published method to detect
the SULT1A1 polymorphism status of each liver (Nowell
et al. 2000; Ozawa et al. 1998). Amplified DNA fragments were
digested with HaeII, and the fragments were resolved on
2% (weight/volume) agarose gels. Fragments from individuals homozygous
for SULT1A1*1 exhibited two bands, visualized by ultraviolet
transillumination, whereas DNA fragments from individuals homozygous
for SULT1A1*2 were not cleaved by this enzyme and exhibited one
band.
Expression and partial purification of SULT isozymes. The
expression of human SULT1A1*1, SULT1A3, SULT1B1, and SULT1E1
in Escherichia coli has been described previously (Dajani
et al. 1998; Wang et al. 1998). We grew E. coli cells
containing the respective sulfotransferase genes as described
previously (Falany et al. 1990, 1994), and prepared 100,000g supernatant
fractions for use in inhibition studies and for partial purification
of the SULT enzymes. We purchased expressed SULT1A1*2 cytosolic
extract from PanVera (Madison, WI) and used it as supplied.
The 100,000g supernatant fractions of SULT1A1*1, SULT1A3,
SULT1B1, and SULT1E1 were partially purified by chromatographic
methods (Falany et al. 1990, 1994). After the last step, a 3'-phosphoadenosine
5'-phosphate (PAP)-agarose affinity column, we dialyzed the fractions
eluted with PAP with three changes of buffer to remove PAP before
the assay of SULT activity with 3-OH-BaP as substrate. We analyzed
active fractions by SDS-PAGE (Laemmli 1970) to assess the purity
of each SULT enzyme. We stained the gels with Coomassie R-250
reagent and determined the percentage of protein present as each
respective SULT enzyme by scanning densitometry.
Kinetic analysis of 3-OH-BaP sulfonation. We
determined SULT activity with 3-OH-BaP as substrate by a fluorimetric
assay of BaP-3-SO4 product formation, as described
previously (Wang et al. 2004). We ensured that the formation
of BaP-3-SO4 was linear for time and protein and did
not exceed 10% of the added 3-OH-BaP with each of the enzyme
sources used. Duplicate tubes were prepared for each incubation
condition. We examined the kinetics of sulfonation in three liver
cytosol fractions by systematically varying the concentration
of 3-OH-BaP or PAPS. When the variable substrate was 3-OH-BaP,
we used 12 concentrations in the range from 0.035 to 2.00 µM,
and the concentration of PAPS was kept constant at 10 µM.
When we varied PAPS, we used 7 concentrations from 0.157 to 10.0 µM
and kept the concentration of 3-OH-BaP constant at 0.100 µM.
We determined the kinetic parameters for 3-OH-BaP sulfonation
by partially purified preparations of the cDNA-expressed SULT
isozymes under incubation conditions similar to those used for
liver cytosol. For SULT1A1*1 and -1A1*2, we used seven substrate
concentrations in the range from 5 to 100 nM; for SULT1E1 we
used six 3-OH-BaP concentrations from 15.6 to 1,000 nM; and for
SULT1A3 and 1B1 we used seven concentrations of 3-OH-BaP from
0.25 to 5.0 µM.
Inhibition of SULT activity by OH-PCBs. To assess
inhibition of 3-OH-BaP SULT activity, we prepared stock solutions
of OH-PCBs in dimethyl sulfoxide (DMSO) and added aliquots to
incubation mixtures such that the final concentration of OH-PCB
was in the range of 0.01-200 µM and the DMSO concentration
did not exceed 0.5% (vol/vol). For each OH-PCB, we examined the
concentration dependence of inhibition with three liver cytosol
fractions, as well as with cytosol fractions from the E. coli expressing
SULT1A1*1, SULT1A3, SULT1B1, and SULT1E1, and the purchased Sf-9
cytosol fraction (PanVera, Madison, WI) containing SULT1A1*2.
For studies with HL cytosol, SULT1A1*1, and SULT1E1, we examined
two concentrations of 3-OH-BaP, 0.1 µM and 1.0 µM.
For studies with SULT1A1*2, we examined only 0.1 µM 3-OH-BaP,
a concentration that did not elicit substrate inhibition. For
studies with SULT1B1, we used only 1.0 µM 3-OH-BaP because
this enzyme had very low activity at 0.1 µM 3-OH-BaP and
did not exhibit substrate inhibition. Examination of the effect
of 50 µM concentrations of several OH-PCBs on the activity
of SULT1A3, measured with 1.0 µM 3-OH-BaP, revealed little
inhibition, so no further concentrations were studied.
Kinetics of inhibition. To study the type of
inhibition produced by OH-PCBs, we used 4´-OH-CB112 (B5)
as a model inhibitor. We prepared four sets of assay tubes containing
HL cytosol and varying amounts of 3-OH-BaP from 35 to 150 nM:
one set (control) contained no 4´-OH-CB112; the other sets
contained 0.25 µM, 0.5 µM, or 1.0 µM 4´-OH-CB112.
Data analysis. We calculated the enzyme kinetic
parameters from studies with variable concentrations of 3-OH-BaP
using nonlinear regression analysis and GraphPad 4.0 software
(GraphPad Software, San Diego, CA). We selected the built-in
Michaelis-Menten equation for most analyses. Where we found evidence
of 3-OH-BaP substrate inhibition, we fit the data into an equation
derived from a two-substrate model (Zhang et al. 1998):
This equation denoted the constant for binding of the first substrate (S) molecule
as Km and the second substrate molecule as Ki. V1 is
the maximum rate for the noninhibitory substrate concentration range, and V2 is
the minimum rate in the inhibitory substrate concentration range
We calculated the effects of OH-PCBs on 3-OH-BaP SULT activity
as percentage inhibition compared with the controls without an
inhibitor. We obtained IC50 values by fitting log
OH-PCB concentration and percent control activity to a sigmoidal
curve. We examined the relationships between IC50 and
physicochemical properties of the OH-PCBs by linear correlation
analysis. We calculated the inhibitory constant (Ki)
from the kinetic studies with 4´-OH-CB112 by means of Dixon
plots and plots of Km/Vmax against
inhibitor concentration (Cornish-Bowden 1995).
SULT1A1 genotype of the liver donors. We
found that the HL cytosols used were from individuals with different SULT1A1 genotypes,
as determined by PCR amplification of the region of the SULT1A gene
flanking the polymorphic base pair. The G to A mutation in SULT1A1 removed
the restriction site for the endonuclease HaeII. As shown
in Figure 2, an individual homozygous for the SULT1A1*2 allele
did not have the HaeII restriction site, and the PCR product
was not cleaved (lane 1). The PCR product from the individual
homozygous for SULT1A1*1 showed complete cleavage by HaeII,
generating two fragments of approximately 100 and 181 bp (lane
3). Enzymatic digestion of the PCR product from the heterozygote
( SULT1A1*1/*2) generated one band of 281 bp and the two
fragments of 100 and 181 bp (lane 2). Thus, the individual liver
designated HL 1 was homozygous for the SULT1A1*1 allele,
HL 2 was heterozygous, and HL 3 was homozygous for the SULT1A1*2 allele.
Sulfonation of 3-OH-BaP by HL cytosol and expressed human
SULT isoforms. Initial studies of the sulfonation
of 3-OH-BaP by HL cytosol revealed that concentrations of
3-OH-BaP > 0.15 µM resulted in a decrease in activity.
To find a saturating concentration of PAPS, we conducted
incubations in the presence of 0.1 µM 3-OH-BaP and
varying concentrations of PAPS. The data fit the Michaelis-Menten
equation, with an apparent Km of 0.56 ± 0.09 µM
and a Vmax of 48 ± 2 pmol/min/mg
protein (mean ± SD; n = 3). The dependence
of activity upon PAPS concentration in expressed human SULT1A1*2,
in the presence of 0.1 µM 3-OH-BaP, also followed Michaelis-Menten
kinetics. The apparent Km was 0.32 µM,
and Vmax was 684 pmol/min/mg protein. As
shown in Figure 3, cytosol and the expressed enzyme were
saturated by a PAPS concentration of 10 µM, and we
used this concentration in subsequent studies.
We conducted detailed studies of the effect of a range of 3-OH-BaP
concentrations up to 2 µM on reaction rates with HL cytosol
and expressed human SULT1A1*2. We obtained preliminary estimates
of the kinetic constants Km and V1 by
fitting the initial rates of sulfonation at concentrations < 0.15 µM
3-OH-BaP to the Michaelis-Menten equation. We then obtained the
values of Ki and V2 through
constraining Km using the equation of Zhang
et al. (1998). We also analyzed data by constraining V1,
but a better fit was found when constraining Km.
Figure 4A shows how the data fit this equation for three individual
HL cytosols. Kinetic studies with expressed SULT1A1*2 revealed
substrate inhibition with the single enzyme (Figure 4B). Table
1 shows values for Km, Ki, V1,
and V2 for each HL cytosol and the expressed
SULT1A1*2. The expressed enzyme showed a lower value for Km (0.022 µM)
and Ki (0.16 µM) than did any of the
HL cytosols.
Table 2 shows the results of kinetic studies with the other
expressed human enzymes. The values shown in Table 2 are from
substrate concentration ranges in which the data fit the Michaelis-Menten
equation. SULT1A1*1 and SULT1E1 showed substrate inhibition at
concentrations of 3-OH-BaP > 0.15 µM, but detailed kinetic
analyses at inhibitory concentrations was not conducted with
these expressed enzymes. We found that SULT1A1*1 had an apparent Km (0.018 µM)
similar to that found with SULT1A1*2 (0.022 µM). SULT1E1
also had high affinity for 3-OH-BaP, with an apparent Km of
0.05 µM. SULT1A3 and SULT1B1 did not exhibit substrate
inhibition over a concentration range up to 5 µM and showed
much higher apparent Km values for 3-OH-BaP.
These expressed enzyme preparations were partially purified,
and SDS-PAGE showed they contained different percentages of the
respective SULT enzymes (Table 2). The values shown for Vmax were
corrected for the percentage of each respective SULT isoform
in the partially purified enzyme preparation.
Inhibition of 3-OH-BaP sulfonation by OH-PCBs with HL
cytosol. The 4-OH-PCBs with one (B group) or two
(C group) flanking chlorine substituents inhibited HL cytosolic
3-OH-BaP sulfotransferase activity in a concentration-dependent
manner. Figure 5A shows inhibition curves from selected OH-PCBs
in the presence of 0.1 µM 3-OH-BaP, and Figure 5B shows
the same compounds studied with 1.0 µM 3-OH-BaP. Table
3 presents the IC50 values of 3-OH-BaP sulfotransferase
activity with all the tested compounds, each at two concentrations
of 3-OH-BaP. Compounds B1-B5 with the 3-chloro-4-hydroxy
substitution pattern were potent inhibitors, with IC50 values
ranging from 0.33 to 1.08 µM, when activity was measured
with 0.1 µM 3-OH-BaP. The OH-PCBs with two chlorine
atoms flanking the hydroxy group (C1-C6) were less potent
inhibitors under these conditions (IC50, 1.31-6.71 µM;
Table 3). The single 6-OH-PCB studied, A1, was a very weak
inhibitor, with an IC50 of > 100 µM (Figure
5). When activity was measured with 1 µM 3-OH-BaP,
a concentration at which substrate inhibition occurred, the
measured IC50 values showed lower inhibitory potencies
for all OH-PCBs, but especially so for the C group compounds,
whose IC50 values ranged from 3 to 58.7 µM
(Table 3).
Inhibition of 3-OH-BaP sulfonation by OH-PCBs with cDNA-expressed
SULTs. For SULT1A1*1, Figure 6A shows inhibition
curves with selected OH-PCBs using 0.1 µM 3-OH-BaP,
whereas Figure 6B shows results with a substrate concentration
of 1.0 µM 3-OH-BaP. We found that 6´-OH-CB35
(A1) was a poor inhibitor of 3-OH-BaP sulfonation under both
conditions of substrate concentration. When using 0.1 µM
3-OH-BaP, type B compounds (B1-B5) showed IC50 values
ranging from 0.77 to 1.31 µM, whereas type C compounds
(C1-C6) exhibited IC50 from 2.16 to 6.65 µM
(Table 3). When using 1.0 µM 3-OH-BaP, the inhibitory
potencies of the OH-PCBs were dramatically reduced. The IC50 values
for type B OH-PCBs were reduced to 10.3-67.5 µM, and
for type C OH-PCBs were 33.8 to > 100 µM (Table
3).
For SULT1A1*2, the IC50 of 6´-OH-CB35 (A1)
was > 100 µM, as shown in Table 3. At 0.1 µM 3-OH-BaP,
the IC50 ranged from 0.54 to 1.48 µM for type
B (B1-B5) compounds and from 1.67 to 6.52 µM for type C
compounds (C1-C6). When using 1.0 µM 3-OH-BaP, the OH-PCB
IC50 was approximately 5 µM for type B (B1-B5)
compounds and 50 µM for type C (C1-C6) compounds (data
not shown).
As shown in Figure 7, expressed SULT1A3 was not inhibited or
was weakly inhibited by OH-PCBs when 3-OH-BaP was used at the
noninhibitory concentration of 1.0 µM. Addition of 50 µM
concentrations of compounds 6´-OH-CB35 (A1), 4´-OH-CB69
(B3), 4´-OH-CB106 (B4), 4´-OH-CB112 (B5), 4´-OH-CB121
(C3), 4´-OH-CB165 (C5), and 4´-OH-CB72 (C6) did not
inhibit the sulfonation of 3-OH-BaP. Compounds 4´-OH-CB35
(B1), 4´-OH-CB36 (B2), 4´-OH-CB79 (C1), and 4´-OH-CB159
(C4) showed 2-20% inhibition at 50 µM, and 4-OH-CB36 (C2)
produced 43% inhibition. Because SULT1A3 activity was poorly
inhibited by 50 µM concentrations, we did not examine a
range of concentrations of OH-PCBs.
Expressed SULT1B1 showed a quite different inhibitory interaction
with OH-PCBs, compared with SULT1A1*1, SULT1A1*2, SULT1A3, and
SULT1E1, in that 6´-OH-CB35 (A1) was a quite potent inhibitor
(IC50, 4.72 µM) of 3-OH-BaP sulfonation (Table
3). Compounds B1 (4´-OH-CB35) and B4 (4´-OH-CB106)
showed IC50 values of 16.76 and 17.45 µM, respectively.
The other type B and type C OH-PCBs were weak inhibitors.
For SULT1E1, compound A1 (6´-OH-CB35) was a poor inhibitor
of 3-OH-BaP sulfonation at either of the substrate concentrations
studied (Table 3). When using 0.1 µM 3-OH-BaP, OH-PCBs
with no or one ortho-substituted chlorine (B1, B2, B4,
C1, C2, C4, and C6) were potent inhibitors of 3-OH-BaP sulfonation,
with IC50 values between 0.24 and 1.3 µM (Table
3). The OH-PCBs with two ortho-substituted chlorine atoms
(B3, B5, C3, and C5) were less potent inhibitors, with IC50 values
of 4.87-7.98 µM (Table 3). When we used 1.0 µM 3-OH-BaP
as substrate, there was a 3- to 5-fold reduction in inhibitory
potency, and the order of potency remained as it was with 0.1 µM
3-OH-BaP.
Structure-activity relationships. For HL cytosol,
expressed SULT1A1*1, SULT1A1*2, and SULT1E1, we investigated
the relationship between inhibitory potency, measured at 0.1 µM
3-OH-BaP, and each of several physicochemical properties of the
4-OH-PCBs. For HL cytosol, SULT1A1*1, and SULT1A1*2, we found
no significant correlation between dihedral angle, molecular
surface area, molecular surface volume, log P, log D at
pH 7.0, or pKa. The IC50 values
with SULT1E1 showed a significant (p < 0.001) linear
correlation with dihedral angle, as shown in Figure 8. No other
significant correlations were found.
Kinetics of 3-OH-BaP sulfotransferase inhibition by 4´-OH-CB112. We
investigated the type of inhibition of 3-OH-BaP sulfonation using
HL cytosol. Figure 9A shows that 4´-OH-CB112 (B5) reduced
sulfotransferase activities at all the tested 3-OH-BaP concentrations
in a concentration-dependent manner. The kinetic constants showed
a steady reduction in Vmax with increasing
concentration of 4´-OH-CB112, but little change in Km,
indicating a noncompetitive type of inhibition (Table 4). Figure
9B shows a plot of Km/Vmax versus
the concentration of 4´-OH-CB112, which indicated a Ki value
for 4´-OH-CB112 of 0.52 ± 0.14 µM.
The major human metabolite of BaP, 3-OH-BaP, was very readily
sulfonated in HL cytosol, especially at concentrations < 0.15 µM.
We observed substrate inhibition in HL cytosol and with SULT1A1
and SULT1E1, but not with SULT1A3 or SULT1B1. We studied the
kinetics of substrate inhibition in liver cytosol and SULT1A1*2
and found that they fit a two-substrate model proposed for the
sulfonation of estradiol by SULT1E1. This model suggested that
SULT1E1 could bind two molecules of estradiol per molecule of
enzyme, one at a preferred site for sulfonation and the other
at an allosteric site associated with substrate inhibition (Zhang
et al. 1998). Our results suggest a similar scenario for the
interaction of 3-OH-BaP with SULT in HL cytosol and SULT1A1.
The Km values for each of the three tested
HL cytosol fractions (48-51 nM), SULT1A1*2 (22 nM), and SULT1A1*1
(18 nM) were low, indicating that 3-OH-BaP has a very high affinity
for human SULT1A1. The Ki values were about
10-fold higher. The 3-OH-BaP was not, however, specific for SULT1A1
but was a substrate for the other human phenol sulfotransferases
studied. In particular SULT1E1 showed a high affinity for 3-OH-BaP,
with a Km of 50 nM. A related compound, 1-hydroxypyrene,
also had a very low Km with SULT1A1 (8 nM)
and SULT1E1 (21 nM) but a higher Km with SULT1A3
(0.8 µM) (Ma et al. 2003). When we calculated 3-OH-BaP
clearance values (Vmax/Km)
for the four partially purified SULT isoforms, the highest value
was found for SULT1A1*1 (Table 2). Thus, 3-OH-BaP was a selective
but not specific substrate for SULT1A1. Other investigators showed
that the SULT1B1 protein content in liver cytosol was about one-fourth
that of SULT1A1 (Honma et al. 2002). The present study showed
that expressed SULT1B1 had a 40-fold higher Km value
(2.0 µM) than found in HL cytosol (0.05 µM), so it
is not likely to contribute much to HL cytosolic sulfonation
of 3-OH-BaP at 0.1 µM substrate concentration (Table 2).
Although SULT1A3 had activity with 3-OH-BaP, it is expressed
at very low levels in the adult liver (Richard et al. 2001) and
is unlikely to contribute much to 3-OH-BaP sulfonation in human
liver. Because Km values for 3-OH-BaP in HL
cytosol were similar to those of purified SULT1A1 and SULT1E1,
and others have shown that SULT1A1 is expressed in liver at approximately
14-fold higher concentrations than SULT1E1 (Honma et al. 2002),
we conclude that the observed activity with 3-OH-BaP in HL cytosol
is catalyzed largely by SULT1A1. Differing structural features
for inhibition of SULT1A1 and SULT1E1 by OH-PCBs further support
our conclusion that, in HL cytosol, activity with 3-OH-BaP is
due primarily to SULT1A1. By chance, the three HL cytosol fractions
we used in these studies were from individuals with different SULT1A1 genotypes.
One was SULT1A1*1 homozygous, a second was heterozygous
for SULT1A1*1/*2, and the third was SULT1A1*2 homozygous.
Kinetic analysis showed little difference among the three cytosol
fractions for V1, which was 121 pmol/min/mg
for the homozygous SULT1A1*1 liver and 94 pmol/min/mg
protein for the SULT1A1*2 liver (Table 1); however, the
small size of our sample precludes a more detailed analysis of
genotype effects on 3-OH-BaP sulfonation activities.
In previous studies, we showed that OH-PCBs inhibited 3-OH-BaP
sulfonation in catfish intestinal cytosol (van den Hurk et al.
2002) and that a compound structurally related to OH-PCBs, 2,4,4´-trichloro-2´-hydroxydiphenyl
ether (triclosan), inhibited sulfonation and glucuronidation
of 3-OH-BaP and other substrates in HL cytosol and with SULT1A1,
SULT1B1, and SULT1E1 (Wang et al. 2004). Here we demonstrated
that a set of 4-OH-PCBs inhibited SULT activity with 3-OH-BaP,
the major metabolite of another pollutant chemical, BaP, in HL
cytosol as well as with cDNA-expressed SULTs. In HL cytosol,
all the 4-OH-PCBs examined inhibited the sulfonation of 3-OH-BaP.
Under incubation conditions in which the 3-OH-BaP substrate did
not cause substrate inhibition (0.1 µM 3-OH-BaP), compounds
with one chlorine atom adjacent to the OH group (B1-B5) were
more potent inhibitors of sulfonation than were compounds in
type C, with chlorine atoms flanking the OH group on each side.
We observed very similar results for potency of inhibition and
order of inhibitory potency with all three liver cytosol fractions
and the two allelic variants of expressed SULT1A1. When incubated
with 1.0 µM 3-OH-BaP, a concentration that produced substrate
inhibition in liver cytosol and with both SULT1A1 variants, the
OH-PCBs were considerably less potent inhibitors in cytosol and
even more so with the expressed SULT1A1*1 and SULT1A1*2 enzymes
(Table 3 and data not shown). The effect of substrate concentration
on the inhibitory potency of the OH-PCBs suggested the possibility
that the OH-PCBs competed with the 3-OH-BaP for an inhibitory
site of the SULT1A1 protein. Gamage et al. (2003) reported that
SULT1A1*2 could accommodate two molecules of the xenobiotic model
substrate p-nitrophenol in the active site. They proposed
that substrate inhibition at high concentrations of p-nitrophenol
was due to impeded catalysis when both binding sites were occupied.
The active site of SULT1A1 appears to be plastic enough to accept
a wide range of hydrophobic phenolic compounds (Gamage et al.
2003) and may be able to accommodate two molecules of 3-OH-BaP,
leading to substrate inhibition, or one molecule of 3-OH-BaP
and one molecule of OH-PCB, resulting in the OH-PCB inhibiting
3-OH-BaP sulfonation. The kinetic studies with 4´-OH-CB112
(B5) showed that the mechanism of inhibition was noncompetitive.
This result could fit the scenario for inhibition discussed above
but does not suggest direct competition of the OH-PCB for binding
to the active site in an orientation that favors sulfonation.
Whatever the mechanism of inhibition, the loss in inhibitory
potency of OH-PCB when assays were conducted with 1.0 µM
3-OH-BaP suggested that the enzyme favored binding of 3-OH-BaP
over binding of OH-PCB, and this was especially true for type
C OH-PCBs, which showed a greater loss in potency than did the
type B compounds. These findings suggest that OH-PCBs are likely
to be poor substrates for sulfonation, but this has not been
studied in human liver.
We could not discern any other clear relationship of inhibitory
potency with structural features or with physicochemical properties
of the OH-PCBs in this relatively small series of compounds,
with cytosol or the two expressed SULT1A1 enzymes. The small
size of the series of compounds studied and the lack of ready
availability of a systematic series of 4-OH-PCBs prevent further
analysis of structure-potency relationships at this time.
Of the other expressed enzymes studied, only SULT1E1 exhibited
potent inhibition by the 4-hydroxylated PCBs. The structure-inhibitory
potency requirement for SULT1E1 was very different from that
for HL cytosol, SULT1A1*1, or 1A1*2, where type B compounds were
more potent inhibitors than were type C OH-PCBs. With SULT1E1,
OH-PCBs with no or one ortho-substituted chlorine were
more potent as inhibitors of 3-OH-BaP sulfonation than were those
with two ortho-substituted chlorine atoms. Substituted
biphenyls with less than one ortho substituent preferentially
adopt coplanar conformation of the two phenyl rings, whereas
those with two or more ortho substituent atoms take on
noncoplanar conformations. We found a significant linear correlation
between inhibitory potency and calculated solution dihedral angles
(Figure 8, Table 3). Similarly, Kester et al. (2000) found that
the best OH-PCB inhibitors of estrogen sulfonation (IC50 values < 5
nM) did not have chlorine substituents at the 2- or the 6-position.
Shevtsov et al. (2003) later showed that 4,4´-di-OH-CB80
(4,4´-di-OH-3,3´,5,5´-tetrachlorobiphenyl)
did not bind the SULT1E1 in a planar conformation, but rather
with a 30° twist between the phenyl rings. We found that
the four OH-PCBs with solution dihedral angles of 38° were
more potent inhibitors than were those with larger dihedral angles.
Although it is possible that interaction with the protein could
alter the conformation of the OH-PCBs, resulting in a different
dihedral angle for the enzyme-bound OH-PCB, our results show
that lack of ortho substituents is associated with higher
inhibitory potency for a xenobiotic SULT1E1 substrate, 3-OH-BaP.
SULT1A3 metabolized 3-OH-BaP with a very high Vmax,
although its preferred substrates are reported to be catecholamines
and other monocyclic phenols containing hydrogen bond donors
(Dajani et al. 1998). Interestingly, 50 µM OH-PCBs caused
little or no inhibition of this enzyme, thereby showing that
the inhibitory interaction was enzyme selective. SULT1B1, the
thyroid hormone sulfotransferase, catalyzed the sulfonation of
3-OH-BaP; however, OH-PCBs that were potent inhibitors of SULT1A1
were only weak inhibitors of the SULT1B1-catalyzed reaction.
In contrast to results with the other enzymes, compound A1 (6´-OH-CB35)
was a fairly potent inhibitor of SULT1B1 (Table 3). Previously, ortho-, meta-,
and para-hydroxylated PCBs were found to inhibit thyroid
hormone sulfonation (Schuur et al. 1998). The meta-hydroxylated
PCB, 3-OH-2,3´,4,4´,5-pentachlorobiphenyl (3-OH-CB118),
was the most potent inhibitor of thyroid hormone sulfonation
in male rat liver cytosol, followed by two para-hydroxylated
PCBs. The ortho-hydroxylated PCB had the lowest potency
among the four OH-PCBs studied. However, with 3-OH-BaP as substrate,
the ortho-OH-PCB, 6´-OH-CB35, was a more potent
inhibitor than were those with para-OH groups, which suggested
that the inhibitory interaction with SULT1B1 was substrate dependent.
Because several OH-PCBs have been detected in human blood and
are presumably also present in liver and other tissues, it is
important to understand their biologic activities. Some OH-PCBs
interact with components of thyroid hormone and estrogen hormone
systems (Kester et al. 2000; Klasson-Wehler et al. 1993; Schuur
et al. 1998; Sinjari and Darnerud 1998). Our finding that OH-PCBs
inhibited the sulfonation of 3-OH-BaP in HL suggests another
aspect of the toxicology of OH-PCBs. The interaction with phenol
sulfotransferase may be of toxicologic importance because sulfonation
is a major pathway of xenobiotic biotransformation (Glatt 2002).
Sulfonation is particularly important at low concentrations of
hydroxylated xenobiotics, such as may be encountered from environmental
exposure to pollutants that require CYP-dependent biotransformation
to introduce a hydroxyl group before their elimination. Formation
of sulfate conjugates of phenolic xenobiotics usually decreases
their toxicity, so inhibition of this pathway may lead to prolonged
exposure to the parent compound, a shift to an alternative phase
II conjugation pathway, glucuronidation, or to further CYP-dependent
metabolism. Both 3-OH-BaP and BaP-3-glucuronide bind to hemoglobin
(Sugihara and James 2003), a potentially toxic interaction. Further
CYP-dependent biotransformation of 3-OH-BaP may lead to more
toxic metabolites such as 3-OH-BaP-7,8-dihydrodiol-9,10-oxide
(Glatt et al. 1987; Ribeiro et al. 1986). On the other hand,
xenobiotics that are activated by sulfonation, such as 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine
(Ozawa et al. 1998), may be rendered less toxic in the presence
of inhibitors of sulfonation.
Our findings may be placed in the context of the structures
of OH-PCBs that have been reported in human blood. All OH-PCB
metabolites identified in blood have the hydroxy group in a para-
or meta-position, with chlorine atoms on vicinal carbon
atoms (Hovander et al. 2002; Sandau et al. 2000, 2002; Sjodin
et al. 2000). The para-OH-PCBs found in blood are likely
to fall into the type C OH-PCBs examined in this study. Although
these were generally less potent as inhibitors of SULT1A1 than
the type B OH-PCBs, it is possible that the concentrations of
these OH-PCBs may reach inhibitory levels in tissues of highly
exposed people or animals. Sjodin et al. (2000) reported total
measured OH-PCB concentrations of up to 6 µM in blood lipids,
whereas Sandau et al. (2000) reported whole blood concentrations
up to 30 nM. Tissue concentrations have not been reported but
they may be higher than blood levels. Type B OH-PCBs with the
3-chloro-4-hydroxy substitution pattern do not appear to be persistent
in blood; however, of the 209 PCB congeners, 19 have a 3-chloro
substitution in one of the phenyl rings, which can be biotransformed
to type B OH-PCBs. If type B OH-PCBs are formed in people, their
high potency as inhibitors of 3-OH-BaP sulfonation may cause
increased toxicity in people who are coexposed to PAH and PCBs.
We found that several OH-PCBs, especially those with a 3-chloro-4-hydroxy
substitution pattern in the phenolic ring, inhibited the sulfonation
of 3-OH-BaP in cytosol and with SULT1A1 at submicromolar concentrations.
Some OH-PCBs with no or one ortho chlorine were potent
inhibitors of 3-OH-BaP sulfonation with SULT1E1. SULT1B1- and
SULT1A3-catalyzed sulfonation of 3-OH-BaP was less sensitive
to inhibition by OH-PCBs. The inhibitory interaction of OH-PCBs
with SULT1A1 and SULT1E1 may have consequences for the biotransformation
and toxicity of phenolic xenobiotics. |
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