Differential Binding Affinities of PCBs, HO-PCBs, and Aroclors with Recombinant Human, Rainbow Trout (Onchorhynkiss mykiss), and Green Anole (Anolis carolinensis) Estrogen Receptors, Using a Semi-High Throughput Competitive Binding Assay

Jason Matthews and Tim Zacharewski1

Department of Biochemistry and National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824–1319

Received September 8, 1999; accepted October 15, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A comparative study was undertaken to assess the ability of 44 polychlorinated biphenyls (PCBs), 9 hydroxylated PCBs (HO-PCBs), and 8 aroclors at concentrations ranging from 1 nM to 10 µM to compete with [3H]17ß-estradiol (E2) for binding to bacterially expressed fusion proteins using a semi-high throughput competitive-binding assay. The fusion proteins consisted of the D, E, and F domains of human ({alpha}), cloned reptilian (Anolis carolinensis) and recloned rainbow trout (Onchorhynkiss mykiss) estrogen receptors (ER) linked to the glutathione S-transferase (GST) protein. GST-hER{alpha}def (human), GST-aERdef (reptile) and GST-rtERdef (rainbow trout) fusion proteins exhibited high affinity for E2 with dissociation constants (Kd) of 0.4 ± 0.1 nM, 0.7 ± 0.2 nM, and 0.6 ± 0.1 nM, respectively. Of the 44 PCBs examined, only PCBs 104, 184, and 188 effectively competed with [3H]E2 for binding to the GST-rtERdef protein with IC50 values ranging from 0.4–1.3 µM. In contrast, these same congeners only caused a 30% displacement of [3H]E2 in GST-hER{alpha}def and GST-aERdef proteins. Several additional congeners were found to bind to the GST-rtERdef fusion protein, although the degree of interaction varied among congeners. Among the HO-PCBs, 2',3',4',5'-tetrachloro-4-biphenylol and 2,6,2',6'-tetrachloro-4-biphenylol bound to all three fusion proteins with IC50 values ranging from 0.1–0.3 µM. Dimethyl sulphoxide (DMSO) concentrations of 20% significantly increased the ability of PCBs 104, 184, and 188 to compete with [3H]E2 for binding to the GST-ERdef fusion proteins, whereas at 20% DMSO, a significant reduction in saturable binding was observed. These results demonstrate that ERs from different species exhibit differential ligand preferences and relative binding affinities for PCBs, which can be dramatically affected by DMSO concentration.

Key Words: polychlorinated biphenyls; estrogenic endocrine disruptors; estrogen receptor; comparative; competitive binding.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In recent years, there has been heightened concern regarding exposure to chemicals in the environment that may interfere with the endocrine system and adversely affect normal reproductive development and fitness of humans and wildlife (McLachlan and Korach, 1995Go). These substances, known as endocrine disrupting chemicals (EDCs), are commonly used throughout modern society. In response, the Safe Drinking Water Act and the Food Quality Protection Act were amended to require the United States Environmental Protection Agency (U.S. EPA) to screen and test for chemicals that mimic or inhibit the activities of estrogen, androgen, and thyroid hormones. The Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) was established and charged with the development and implementation of a screening and testing strategy (EDSTAC, 1998Go). EDSTAC deliberations resulted in a tiered strategy consisting of prioritization followed by screening and testing approaches that involve a combination of in vitro and in vivo assays. The U.S. EPA is currently exploring the possibility of using high-throughput receptor binding and reporter gene assays to assist in prioritizing chemicals that require testing.

Much attention has been focused on estrogenic endocrine disruptors (EEDs). These chemicals encompass a wide range of substances including natural products, environmental pollutants, pharmaceuticals, and industrial chemicals (Colborn, 1993; Katzenellenbogen, 1995Go). Many of these chemicals do not share any obvious structural similarity to the endogenous ligand for the estrogen receptor (ER), 17ß-estradiol (E2), which makes identification based solely on molecular structure difficult (Katzenellenbogen, 1995Go). It has been hypothesized that many of the effects elicited by estrogenic substances are the result of ER-mediated modulation of gene expression (McLachlan, 1993Go), although additional modes of action cannot be discounted.

The ER is a member of the nuclear receptor superfamily, a family of nuclear proteins that function as transcription factors to modulate gene expression in a ligand-dependent manner (Evans, 1988Go). As with other members of this family, the ER has a modular structure consisting of six domains (A–F) (Evans, 1988Go; Tenbaum and Baniahmad, 1997Go). The highly conserved DNA-binding domain (C domain) separates a highly variable NH2-terminal region (A/B domains) and a COOH-terminal ligand-binding domain (D, E, and F domains). The ER and other steroid hormone receptors are activated by interaction with specific ligands that bind with high affinity to the ligand-binding domain. Ligand-occupied ERs undergo homodimerization, and the resulting complex binds to its cognate DNA target site. These sites are referred to as estrogen responsive elements (EREs) and are located in the regulatory region of estrogen-inducible genes. Once bound to the ERE, the ER-homodimer complex may induce or inhibit gene transcription, thereby altering the levels of proteins that are important for development, cell proliferation, and the maintenance of homeostasis. Consequently, the ER acts as the primary gatekeeper for initiation of a number of estrogenic responses.

Even though the physiological actions of the ER are conserved among different species, amino acid sequences of ligand binding domains are variable. This suggests that species may exhibit different responses and sensitivities to EEDs, and that one species may not be an appropriate surrogate for use in identifying and predicting responses in other species. A number of competitive-binding studies have shown that EEDs exhibit differential binding preferences and relative binding affinities for the ER of different species (Connor et al., 1997Go; Fitzpatrik et al., 1989; Le Drean et al., 1995Go; Vonier et al., 1997Go).

Polychlorinated biphenyls (PCBs) are a class of halogenated aromatic industrial compounds that are ubiquitous, persistent environmental contaminants detected in almost every ecosystem (Bellschmiter et al., 1981Go). They were commercially manufactured as mixtures (e.g. Aroclors) containing varying degrees of chlorination made up of 140–150 of the 209 possible congeners (Mullin et al., 1984Go; Safe, 1993Go; Schulz et al., 1989Go). PCBs evoke or elicit a number of in vitro and in vivo responses. PCBs, and hydroxylated (HO)-PCBs have been identified in wildlife and humans, and they represent a class of EEDs that differs significantly from the endogenous ER ligand, E2. Co-planar congeners and their planar, mono-ortho substituted derivatives induce responses that correlate with their binding affinity for the aryl hydrocarbon receptor (AhR) and evoke 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-like responses (Safe, 1993Go). Non-coplanar (di-, tri- and tetra-ortho substituted) PCBs and some HO-PCBs induce multiple responses (Narasimhan et al., 1991Go; Schuur et al., 1998Go; Van den Berg et al., 1991Go) including in vitro and in vivo estrogenic activities independent of the AhR (Bitman and Cecil, 1970Go; Fielden et al., 1997Go; Li et al., 1994Go).

In order to investigate the ability of PCBs to compete with E2 for binding to the ER and to identify potential differences in ER binding among species, a comparative study was undertaken in which a semi-high throughput competitive-binding assay, using bacterially expressed GST-ER fusion proteins, was developed. In this study, 44 PCB congeners, 8 commercial Aroclor mixtures, and 9 HO-PCBs, 7 of which have been detected in human serum (Bergman et al., 1994Go; Moore et al., 1997Go) were examined for their ability to compete with [3H]E2 for binding to the recombinant ERs from human, green anole (Anolis carolinensis) and rainbow trout (Onchorhynkiss mykiss).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Polychlorinated biphenyls (PCB) International Union of Pure and Applied Chemistry (IUPAC) # 41, 51, 58, 60, 68, 78, 91, 99, 104, 112, 115, 126, 143, 153, 169, 173, 184, 188, 190, and 193 were purchased from AccuStandard (New Haven, CT) and provided by M. Tysklind (Umeå University, Umeå, Sweden). PCBs IUPAC # 18, 44, 45, 47, 54, 70, 74, 84, 87, 101, 128, 138, 151, 158, 168, 177, 178, 183, 187, and 194; Aroclors 1016, 1221, 1232, 1242, 1248, 1254, 1260, and 1268 were synthesized or provided by S. Safe (Texas A&M University, College Station, TX). Hydroxylated PCBs 2,3,5,3',4'-pentachloro-4-biphenylol; 2,3,5,2',4',5'-hexachloro-4-biphenylol; 2,4,5,2',3',4',5''-heptachloro-3-biphenylol; 3,5,2',3',4'-pentachloro-4-biphenylol; 2,3,5,2',3',4'-hexachloro-4-biphenylol; 2,3,5,2',3',4',5'-heptachloro-4-biphenylol; 2,3,5,6,2',4',5',-heptachloro-4-biphenylol; 2,6,2',6'-tetrachloro-4-biphenylol; and 2',3',4',5'-tetrachloro-4-biphenylol were synthesized by S. Safe (Connor et al., 1997Go; Safe et al., 1995Go). Dimethyl sulphoxide (DMSO) and 17ß-estradiol (E2) were obtained from Sigma (St. Louis, MO). [2,4,6,7,16,17–3H] 17ß-estradiol ([3H]E2; 123 Ci/mmol) was purchased from New England Nuclear (Boston, MA). MicroScint 20 was obtained from Packard Instruments (Meriden, CT). Isopropylthio-ß-D-galactoside (IPTG) and bovine serum albumin (BSA) were obtained from Fisher Scientific (Pittsburgh, PA). The glutathione S-transferase (GST) expression vector, pGEX6p3, glutathione (GSH) sepharose and XK16 columns were purchased from Amersham/Pharmacia (Piscataway, NJ). SuperScript II reverse transcriptase and Trizol Reagent were purchased from Life Technologies (Gaithesburg, MD). Vent DNA polymerase was purchased from New England Biolabs (Beverly, MA), and restriction enzymes and Taq DNA polymerase were obtained from Roche/Boehringer Mannheim (Indianapolis, IN). All other chemicals and biochemicals were of the highest quality available from commercial sources.

RNA isolation.
Total RNA from a 1 cm3 liver section from a female green anole (Anolis carolinensis; kindly provided by J. Wade, Departments of Psychology and Zoology, Michigan State University) was isolated using Trizol Reagent. The Trizol Reagent procedure is a modification of the single step-RNA isolation method developed by Chomczynski and Sacchi, (1987). Green anole liver sections were homogenized in the presence of Trizol Reagent, using a Brinkman Polytron homogenizer. Following a 5-min incubation at ambient temperature, chloroform was added and the mixture was separated by centrifugation at 12,000 x g for 15 min at 4°C. The aqueous layer containing the isolated RNA was removed and the RNA was precipitated using isopropanol. The RNA was pelleted by centrifugation at 12,000 x g for 10 min at 4°C, and the resulting pellet was washed with 75% ethanol diluted with diethyl pyrocarbonate (DEPC)-treated sterile water. The pellet was then air dried and resuspended in DEPC-treated sterile water. RNA was stored at –80°C until use.

Cloning of green anole estrogen receptor DEF domain.
Total RNA (5 µg) was incubated for 10 min at 70°C with 500 nM oligo dT primer (PR1r). Following a 5 min incubation on ice, the mRNA was reverse transcribed in a 20 µl reaction mixture containing PCR buffer (20 mM Tris–HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl2), 10 mM dithiothreitol (DTT), 500 µM dNTPs and 200 units of SuperScript II reverse transcriptase at 42°C for 50 min. The reaction was terminated with a 15 min incubation at 70°C. The reverse transcription (RT) reaction was then incubated with 1 unit of RNase H for 30 min at 37°C. One tenth of the RT reaction was used in the subsequent PCR reactions.

RACE (rapid amplification of cDNA ends) PCR reactions were performed according to the manufacturer's instructions (Life Technologies). Three degenerate primers were used in the cloning strategy. The oligonucleotides were identified from a consensus sequence derived by a multiple sequence alignment of the ERs from 10 different species. Two of the primers (PR3f and PR4f; see Table 1Go) were based on the highly conserved ER DNA binding domain and the third primer, PR5r, was derived from a highly conserved region in the ligand binding domain (LBD). Optimal PCR reaction conditions were determined to be 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2, 200 nM of each primer, 200 µM dNTPs and 2.5 units of Taq polymerase. Following the addition of template, the samples were incubated at 95°C for 2 min and amplified for 35 cycles. Each cycle included: 1 min denaturation at 95°C, 1 min annealing at 62°C and 2 min elongation at 72°C. Ten percent of the first strand synthesis reaction was PCR amplified using primers PR3f and PR2r. Using a nested PCR strategy, a 2 µl aliquot of the initial PCR reaction was used as a template for a subsequent PCR amplification using primers PR4f and PR2r. The products from the second round of PCR were used as a template in a third PCR reaction using primers PR4f and PR5r. This reaction produced a fragment of approximately 800 bp, which was digested with BamHI and XhoI, cloned into the eukaryotic expression vector, pTL1 and sequenced using ABI/Prism automated sequencing (Perkin Elmer Applied Biosystems; Foster City, CA). Based on this sequence, a green anole ER-specific primer (PR6f) was designed and used with PR2r to amplify the 3' end of the ER using a 2 µl aliquot of the PR4f/PR2r PCR reaction as template. The resulting 1100-bp product was cloned into pGEM plasmid (Promega) and sequenced using ABI/Prism automated sequencing. The boundaries of the green anole ER D, E and F domains were determined by sequence alignment to the human ER{alpha}. Sequence analysis was performed using MacVector 6.5 and the GCG Wisconsin Package (Oxford Molecular Ltd., Beaverton OR).


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TABLE 1 PCR Primers Used in the Cloning and Recloning of the Green Anole and Rainbow Trout Estrogen Receptor (ER) D, E and F Domains and in the Construction of the GST-ERdef Containing Plasmids
 
Recloning of rainbow trout ER DEF domains.
Total RNA (1 µg) from the liver of a female rainbow trout (Onchorhynkus mykiss; kindly provided by S. Wagner, Department of Physiology, University of Western Ontario) was reverse transcribed as previously described (Gillesby and Zacharewski, 1999Go) in the presence of first-strand buffer (50 mM Tris–HCl (pH 8.3), 37.5 mM KCl, 1.5 mM MgCl2), 10 mM DTT, 500 nM reverse primer (PR14r), 1 mM dNTPs, and 40 units of SuperScript II. The RT reaction was incubated for 10 min at 25°C, followed by 50 min at 42°C, 15 min at 70°C and 5 min at 4°C. The entire cDNA product produced in the RT reaction was used in subsequent PCR reactions. The PCR reaction mixture, containing Thermopol buffer (20 mM Tris–HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, and 0.1% Triton X-100), 200 µM dNTPs, 1 mM MgSO4, 2 µM primers (PR13f/PR14r), and 1.25 units of Vent DNA polymerase was amplified for 30 cycles using the following conditions: 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min.

Construction of GST-ERdef fusion proteins.
The plasmids pGEXhER{alpha}def, pGEXaERdef, and pGEXrtERdef were constructed by PCR amplification of the human ER{alpha} (kindly provided by P. Chambon, INSERM U184, Strasbourg, France), green anole, and rainbow trout ER DEF domains using primers PR7f/PR8r, PR9f/PR10r, and PR11f/PR12r, respectively. The fragments were digested with the appropriate restriction enzymes (see Table 1Go) and ligated into the GST fusion protein expression vector, pGEX6p3. The PCR amplification was performed using Vent DNA polymerase (New England Biolabs) as described above. The PCR reaction mixture containing Thermopol buffer, 200 µM dNTPs, 1 mM MgSO4, 500 nM primer, and 1.25 units of polymerase was heated to 94°C for 5 min followed by 35 rounds of 94°C for 45 s, 60°C for 45 s, and 72°C for 1 min 45 s. The sequence of each construct was confirmed with restriction enzyme digest and ABI/Prism automated sequencing.

Expression and purification of GST ER fusion proteins.
Overnight cultures of E. coli strain BL21 (Amersham/Pharmacia) containing pGEX-ERdef constructs were diluted 1:100 in 500 ml of LB broth (1% (w/v) tryptone, 0.5% (w/v) yeast extract, and 1% (w/v) NaCl, pH 7.5) containing 100 µg ampicillin/ml and incubated at 37°C with constant shaking. The cells were grown to an optical density of approximately 1.0 at 600 nm, and induced with IPTG at a final concentration of 1 mM. The induced cultures were incubated for 4 h at 37°C, then pelleted by centrifugation at 1000 x g for 10 min at 4°C. Cell pellets were resuspended in 25 ml of buffer A (50 mM HEPES, 3 mM EDTA, 5 mM DTT, 50 mM NaCl, and 10% (v/v) glycerol, pH 7.5) containing 0.1 mg/ml lysozyme, 100 µg/ml phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin,and 10 µg/ml pepstatin A. Cells were then lysed by sonication on ice for 3 x 15 s, separated by 10 s intervals. Tween20, to a final concentration of 0.1%, was added to the cellular debris and incubated for 30 min at 4°C under constant shaking. Cell debris was pelleted by centrifugation at 20,000 x g for 40 min at 4°C. Supernatants were stored at –80°C until further use.

The supernatants containing the GST fusion proteins were applied to an XK16 column containing GSH sepharose pre-equilibrated with buffer A at a constant flow rate of 0.5 ml/min at 4°C. After adsorption of the protein, the GSH sepharose was washed with 100 ml of buffer B (50 mM HEPES, 3 mM EDTA, 5 mM DTT, 150 mM NaCl and 10% (v/v) glycerol, pH 7.5). Bound proteins were eluted in 25 ml of buffer C (50 mM HEPES, 3 mM EDTA, 5 mM DTT, 150 mM NaCl and 10% (v/v) glycerol, pH 8.0) containing 10 mM GSH. The partially purified protein was concentrated to a 1-ml final volume using Millipore Ultrafree-15 filter columns with a 50-kDa molecular weight cutoff (Millipore Corp., Bedford MA). Protein concentration was determined using the Bradford (1976) method. Protein was diluted to 0.5 mg/ml and stored at –80°C until further use. Partially purified fusion proteins were separated by SDS–PAGE according to Laemmli (1970), using a 4% stacking and 10% separating gel. Proteins were visualized by Coomassie brilliant blue R250 staining.

Receptor binding assays.
Partially purified GST-ERdef fusion proteins were diluted in TEGD buffer (10 mM Tris pH 7.6, 1.5 mM EDTA, 1 mM DTT and 10% (v/v) glycerol) containing 1 mg/ml bovine serum albumin (BSA) as a carrier protein, and incubated at 4°C for 2 h with 0.1–3.5 nM [3H]E2 in 1 ml glass tubes arranged in a 96-well format (Marsh Scientific, Rochester, NY). Fusion protein preparations were diluted to ensure 10,000 dpms of total binding (dilutions varied from 750–3000-fold). Binding assays were initiated by adding 240 µl of protein preparation to glass tubes containing 5 µl of DMSO and 5 µl of [3H]E2; thus, the solvent concentration did not exceed 4%, unless stated otherwise. The amount of nonspecific binding was determined in the presence of a 400-fold excess of unlabeled E2. Bound [3H]E2 was separated from free using a 96-well filter plate and vacuum pump harvester (Packard Instruments). Filter plates containing the protein were washed with 3 x 50 ml of TEG (10 mM Tris buffer (pH 7.6), 1.5 mM EDTA, and 10% (v/v) glycerol) and allowed to dry under continued suction for 30 s. After drying, the underside of the filter plates were sealed and 50 µl of MicroScint 20 scintillation cocktail was added to each well. Bound [3H]E2 was measured using a TopCount luminescence and scintillation counter (Packard Instruments).

Competitive-ligand-binding assays were performed essentially as described above with the following modifications. Partially purified GST-ERdef fusion protein was diluted in TEGD containing 1 mg/ml BSA and was incubated with 2.5 nM [3H]E2 (5 µl aliquot) and increasing concentrations of unlabeled competitor. PCB (1.0 nM–10 µM, 5 µl aliquots) at 4°C for 2 h. Bound [3H]E2 was separated from free as described above. Nonspecific binding was determined in the presence of a 400-fold excess of unlabeled E2. Each treatment was performed in quadruplicate and results are expressed as percent specific binding of [3H]E2 versus log of competitor concentration. IC50 values were determined from nonlinear regression for single site competitive binding analysis, using Equation 1.

The reported IC50 values represent the concentration of test compound required to displace 50% [3H]E2 from the GST-ER fusion proteins as compared to the 50% displacement of [3H]E2 achieved by unlabeled E2. These analyses were performed using GraphPad Prism 3.0 software (GraphPad Software Inc., San Diego, CA).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of Green Anole ER DEF cDNA
Figure 1Go shows the partial nucleotide sequence of the green anole ER (aER). The region spanning the D, E and F domains was of similar size (330 amino acids) to that of the human ER{alpha} (331 amino acids). Boundaries of the individual D, E and F domains and the percent amino acid sequence identity and similarity of the combined and individual domains were deter-mined from a primary sequence alignment to the human ER{alpha} (hER{alpha}) sequence (Fig. 2Go). Combined, the D, E and F domains are 79% identical and 83% similar in terms of amino acid composition to hER{alpha}. The E domain was the most conserved (89% sequence identity and 92% sequence similarity) while the D (57% sequence identity) and F (32% sequence identity) domains were less conserved.



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FIG. 1. Partial nucleotide sequence of the green anole ER. The first amino acid of the D domain is underlined. The boundaries of the domains were determined from an alignment to human ER{alpha}. The numbers on the left indicate the nucleotide position, and the values on the right refer to the amino acids (GenBank accession number AF095911).

 


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FIG. 2. A comparison of the amino acid sequence of human, green anole, and recloned rainbow trout ER D, E, and F domains. Identical residues are shaded in dark gray, similar amino acids in terms of residue type are lightly shaded, and non-conservative changes are not shaded. The boundaries of the individual domains were determined from an alignment to the human ER{alpha}. The numbers flanking each sequence refer to the amino acid residues. The alignment was generated using the ClustalW alignment in MacVector 6.5 (Oxford Molecular Ltd., Beaverton, OR). aOnly a portion of the green anole ER sequence was cloned and the start of the D domain corresponds to amino acid residue 19 in the cloned sequence. bRefers to the first amino acid of the recloned rainbow trout ER partial sequence (GenBank accession number AF099079).

 
Recloning of Rainbow Trout ER DEF cDNA
The rainbow trout ER (rtER) D, E, and F domains were recloned using primers based on the previously published sequence (Pakdel et al., 1990Go). Upon sequencing the fragment, significant differences with the published sequence were observed (Table 2Go), and these differences were confirmed by sequencing 7 independent clones from 3 different animals (data not shown). The recloned sequence (GenBank accession AF099079) is in agreement with 2 unpublished rtER sequences recently deposited in the GenBank database (GenBank accession AF242740 and AF242741) by the authors, who originally published the rtER sequence (Table 2Go). Combined, the D, E, and F domains are 40% identical and 47% similar in terms of amino acid composition, to the hER{alpha}. The E domain was the most conserved (60% sequence identity and 66% sequence similarity) while the D and F domains were less conserved (18% and 19% sequence identity, respectively).


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TABLE 2 Comparison of the Amino Acid Sequences from the D, E, and F Domains of Estrogen Receptor from Rainbow Trout Taken from Four Different Sources. Boundaries of the D, E and F Domains Were Determined from a Sequence Alignment to the Human ER{alpha}
 
Purification of GST-ERdef Fusion Proteins
The purity of the GST-ERdef fusion proteins was qualitatively determined to be approximately 85%, based on a Coomassie-stained SDS–PAGE analysis (Fig. 3BGo). All 3 fusion proteins migrated according to their predicted molecular weights (mw): GST-hER{alpha}def (64.2 kDa), GST-aERdef protein (64.3 kDa), and GST-rtERdef (65.5 kDa). The recovery of the fusion proteins varied among species, with yields ranging from 1–3 mg/l. As shown in Figure 3BGo, lane I, the GST-hER{alpha}def fusion migrated as a doublet. The higher MW band most likely represents the full length product, whereas the lower band may result from proteolytic cleavage. Although the GST-aERdef and GST-rtERdef fusions did not appear to migrate as doublets, additional higher and lower MW proteins co-purified with the fusion proteins.



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FIG. 3. (A) A schematic representation of the GST-ER fusion proteins expressed in bacteria. Amino acid residues that link the 2 proteins are provided above the transition point. The residues shown in italics represent glutathione S-transferase (GST). The first 3 residues of the ER ligand binding domain (DEF domains) are shown in regular text. Numbers provided above identify the amino acids used in the construction. The numbers within the domains represent % identity while those in parentheses represent % similarity to hER{alpha}. a Only a portion of the green anole ER sequence was cloned, and the start of the D domain corresponds to amino acid residue 19 in the cloned sequence. b Refers to the first amino acid of the recloned rainbow trout ER partial sequence (GenBank accession number AF099079). (B) SDS–PAGE analysis of the GST-ERdef fusion proteins purified using GSH affinity chromatography. Lane A: 5 µg of partially purified GST-hER{alpha}def (predicted mw = 64.2 kDa. Lane B: 5 µg of partially purified GST-aERdef (predicted mw = 64.3 kDa. Lane C: 5 µg of partially purified GST-rtERdef (predicted mw = 65.5 kDa). Proteins were analyzed using a 4% stacking and a 12% separating gel stained with Coomassie brilliant blue R250.

 
Characterization of the Purified GST-ERdef Fusion Proteins
Binding affinities of the partially purified GST-ERdef fusion proteins for E2 were determined by saturation analysis (inset, Fig. 4Go) and linear transformation of the data (Scatchard, 1949Go) (Fig. 4Go). Differences in the amounts of receptor required to attain the desired 10,000 dpm at saturation were species-dependent and may be due, in part, to differences in protein purity, functionality, and level of expression between preparations. All GST-ERdef fusion proteins exhibited high binding affinity for E2, with dissociation constants (Kd) of 0.4 ± 0.1 nM, 0.7 ± 0.2 nM, and 0.6 ± 0.1 nM for GST-hER{alpha}def, GST-aERdef and GST-rtERdef, respectively. These values are means ± standard deviations from 4 independent experiments.



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FIG. 4. Saturation analysis of (A) GST-hER{alpha}def, (B) GST-aERdef, and (C) GST-rtERdef fusion proteins. Various concentrations of [3H]E2 were incubated with known amounts of partially purified fusion protein for 2 h at 4°C as described in Materials and Methods. Saturation data shown in the inset were plotted by the method of Scatchard. The reported Kd values were averaged from 4 independent experiments.

 
Ligand Binding Analysis
A set of 44 PCBs, 8 Aroclors, and 9 HO-PCBs were examined for their ability to compete with [3H]E2 for binding to the GST-ERdef fusion proteins using a semi-high throughput competitive-binding assay. Protein preparations were incubated with 2.5 nM [3H]E2 and increasing concentrations of PCBs (1.0 nM- 10 µM) for 2 h at 4°C. Kinetic studies with E2 showed that a 2-h incubation was sufficient to reach full saturation (data not shown).

Figure 5Go shows binding profiles of 4 representative PCB congeners to the GST-rtERdef fusion protein, and illustrates the criteria used to classify competitive binding. The binding patterns observed for PCB 77 and PCB 153 represent PCB congeners that are classified as non-binders (nb) and weak binders (wb), respectively. A PCB congener was classified as a non-binder if less than 10% competitive binding was observed; similarly, a PCB congener was classified as a weak binder if only 10%-50% of [3H]E2 was displaced at the highest concentration of competitor examined (10 µM). PCB 91 effectively displaced 50%-70% [3H]E2 from the GST-rtERdef, however, a characteristic one-site competitive displacement curve was not achieved. Consequently, an IC50 greater than the highest concentration of test compound was ascribed. PCB 184 effectively competed with [3H]E2, displacing more than 80% [3H]E2 from the fusion protein and an IC50 value was calculated using Graphpad Prism 3.0. Concentrations greater than 10 µM were not examined, due to potential solubility limitations of the test compounds.



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FIG. 5. Classification of PCB interactions with GST-ERdef fusion proteins. An aliquot of partially purified GST-rtERdef was incubated with 2.5 nM [3H]E2 and increasing concentrations of unlabeled competitor [filled circle] E2 (positive control)—exhibits full displacement curve with IC50 2.4 ± 1.0 nM, [empty square] PCB 77—non-binder (nb), did not compete with [3H]E2 for binding to GST-ERdef fusion protein, [filled square] PCB 153—weak binder (wb), only 10–30% displacement of [3H]E2 from GST-ERdef fusion protein was observed at the highest dose examined, x PCB 91—congener capable of displacing at least 50% [3H]E2 at the highest examined dose examined from GST-ERdef fusion proteins however characteristic displacement was not achieved IC50 > 10 µM, and [empty circle] PCB 184 exhibits full displacement curve with EC50 = 3.1 ± 0.6 nM. The results (mean and standard deviation) are from a representative experiment that was repeated 3 times.

 
Table 3Go summarizes the competitive binding ability of 44 PCB congeners to GST-hER{alpha}def, GST-aERdef and GST-rtERdef fusion proteins. Only PCBs 104, 184, and 188 competed with [3H]E2 for binding to GST-hER{alpha}def and GST-aERdef fusion proteins. The remaining compounds did not significantly bind to either fusion protein.


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TABLE 3 Summary of the Ability of 44 Polychlorinated Biphenyls to Compete with [3H]17ß-Estradiol for Binding to Recombinant Fusion Proteins Containing the Human, Green Anole and Rainbow Trout Estrogen Receptor D, E, and F Domains
 
In contrast, several PCBs competitively bound to GST-rtERdef. All 5 mono-ortho-substituted PCBs (PCB 58, 60, 68, 70, and 74) weakly interacted with GST-rtERdef fusion protein and displaced less than 30% of the radioligand. Nine of the 18 di-ortho-substituted congeners (PCB 18, 44, 49, 99, 101, 112, 128, 138, and 153) exhibited weak competitive binding profiles, while PCB 41, 47, and 115 displaced between 50 and 70% [3H]E2 from the GST-rtERdef fusion protein. Of the 13 tri-ortho-substituted PCBs examined, 7 congeners (PCB 84, 95, 149, 151, 178, 183, and 187) weakly competed with [3H]E2, and 6 congeners (PCB 45, 51, 91, 143, 173, and 177) displaced between 50 and 70% of [3H]E2 from the GST-rtERdef fusion protein. The most active congeners contained 4 ortho-chloro substituents; PCB 104, 184, and 188 were strong binders, while PCB 54 only displaced 50–70% [3H]E2 from the fusion protein. None of the 4 co-planar (non-ortho substituted) PCBs examined effectively competed with [3H]E2 for binding, with the exception of PCB 169, which demonstrated a weak interaction with the GST-rtERdef fusion protein. The differential binding of PCB 47 to the GST-ER fusion proteins is shown in Figure 6Go. PCBs 41, 45, 51, and 91 showed similar binding curves across the species (data not shown).



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FIG. 6. Representative competitive binding curves for [filled circle] E2 to GST-hER{alpha}def fusion protein and unlabeled PCB 47 with [empty circle] GST-rtERdef, [filled square] GST-hER{alpha}def and [empty square] GST-aERdef fusion proteins. An aliquot of partially purified GST-ERdef fusion proteins was incubated with 2.5 nM [3H]E2 and increasing concentrations of unlabeled E2 or PCB 47 as described in Materials and Methods. The results (mean and standard deviation) are from a representative experiment that was repeated 3 times.

 
Results in Table 4Go summarize the competitive binding of 9 hydroxylated PCB congeners to GST-ERdef fusion proteins. HO-PCB X and HO-PCB 54 competitively bound to all 3 fusion proteins with similar affinity (IC50 values 0.1–0.3 µM). None of the 8 Aroclor mixtures competitively bound to any of the GST-ER fusion proteins at the highest concentration tested (10 µM). Higher concentrations were not examined due to possible solubility limitations.


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TABLE 4 Receptor Binding Activity of 9 Hydroxylated Polychlorinated Biphenyls to Each of the Recombinant Fusion Proteins Containing the Human, Green Anole, and Rainbow Trout Estrogen Receptor D, E, and F Domains
 
Table 5Go summarizes effects of DMSO on competitive binding of E2 and PCB to GST-ERdef fusion proteins. IC50 values for E2 were unchanged at DMSO concentrations up to 20% v/v in the assay buffer. In contrast, DMSO increased the binding affinity of select PCB congeners for the fusion proteins. Figure 7AGo shows the effect of DMSO on the competitive binding of E2 and PCB 184 to GST-hER{alpha}def. At 4% DMSO, PCB 184 displayed characteristics of a weak binder; however, as the DMSO concentration increased to 20%, PCB 184 was a more effective competitor for binding to GST-hER{alpha}def.


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TABLE 5 Effects of Dimethyl Sulphoxide on the Ability of Select Polychlorinated Biphenyls to Competitively Displace [3H]17ß-Estradiol from Recombinant Fusion Proteins Containing the Human, Green Anole, and Rainbow Trout Estrogen Receptor D, E, and F Domains
 


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FIG. 7. (A) Competitive binding of E2 and PCB 184 at 4%, 10%, and 20% DMSO in assay buffer. (B) The effect of increasing DMSO concentration on the binding of [3H]E2 to the GST-ER DEF fusion protein. A 240 µL aliquot of the GST-hER{alpha}def (H), GST-aERdef (A) or GST-rtERdef (Rt) fusion proteins were dissolved in TEG buffer, pH 7.6 containing 1 mg/ml BSA as a carrier protein, and incubated with 2.5 nM [3H]E2 and the indicated final concentration of DMSO and unlabeled competitor as indicated, for 2 h at 4°C. The amount of bound radionucleotide was compared within each species and among different concentrations of DMSO. The results (mean and standard deviation) are from a representative experiment that was repeated two times. * Indicates DMSO treatment significantly different compared to other concentrations of DMSO for each fusion protein (p < 0.05) using a paired Student's t-test.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recombinantly expressed human, green anole, and rainbow trout ERs were used in this study to systematically investigate the potential differences in the ligand-binding preference of structurally diverse PCBs and HO-PCBs among ERs from different species. The human ER{alpha} (hER{alpha}), considered to be the prototypical ER, was selected as the basis for all comparisons, due to the information available on its ligand-binding characteristics and the structure of the ligand-binding pocket (Brzozowski et al., 1997Go; Tanenbaum et al., 1998Go). The rainbow trout (Onchorhynkus mykiss) ER (rtER) was included in the study, since it represents an environmentally relevant species, and the rtER has the most divergent amino acid sequence within its ligand-binding domains (LBD, domains D, E, and F) of any cloned ER with percent identity and similarity of 40 and 47%, respectively, when compared to the hER{alpha} LBD. The ER from the green anole (Anolis carolinensis), a lizard commonly found throughout the southeastern United States, was also included to further investigate claims of unexpected interactions between reptilian ERs and xenobiotics (Crain et al., 1998Go; Crews et al., 1995Go; Vonier et al., 1996Go). The green anole ER (aER) represents the first reported complete LBD sequence for a reptile, although partial sequences have been reported (Bergeron et al., 1998Go; Young et al., 1995Go). The LBD of aER is intermediate in amino acid sequence divergence relative to hER{alpha} and rtER LBD, with percent identity and similarity of 79 and 83%, respectively, compared to hER{alpha}. These divergent sequences were selected in order to assess the appropriateness of using a single surrogate species to prioritize, identify, and assess potential EEDs. Moreover, analysis of the data and comparison of ER sequence information may identify amino acid residues that might contribute to differences in ligand preference and relative binding affinities among species.

The D, E, and F domains of the rtER were recloned and contained several differences when compared to the originally published sequence (Table 2Go). A single cytosine inserted at nucleotide position 726 of the originally published sequence (Pakdel et al., 1990Go) resulted in a shift in the reading frame. This change was later corrected by a second insertion of a pair of cytosines at nucleotide positions 756 and 757 (Pakdel et al., 1990Go). The insertion caused a change of 9 amino acids and resulted in the addition of a single amino acid residue within the insertion sites (Table 2Go). Moreover, single amino acid changes were found at various sites within the D, E, and F sequences (Table 2Go). It is doubtful that our sequence represents a polymorphism since the same changes were reported in two recently submitted rtER sequences in the GenBank database (GenBank accession AF242740 and AF242741).

Potential species-specific sensitivities to PCBs were investigated using the ER D, E, and F domains from mammalian (human), reptilian (green anole),and fish (rainbow trout) species expressed in bacteria as GST fusion proteins, using a semi-high throughput competitive binding assay. In vitro ER competitive binding assays have been well established and extensively used to investigate ER-ligand interactions. All competitive binding assays involve the displacement of a receptor-bound probe molecule by a test compound. The probe is usually [3H]E2; however, fluorescently labeled high-affinity ER ligands have also been used (Bolger et al., 1998Go). Separation of receptor-bound from free ligand can be done using dextran-coated charcoal (DCC) (Stoessel and Leclercq, 1986Go), hydroxyapatite (HAP) (Laws et al., 1996Go) or by protein binding to a glass fiber membrane (Coleman et al., 1997Go). Shortcomings of the assay include the inability to distinguish between receptor agonists and antagonists and the possibility that high concentrations of competing ligand may lead to an increase in non-specific binding (Zacharewski, 1997Go). However, the assay is amenable to a high throughput format and can be used to investigate direct ligand:ER interactions, which are the initial steps in many estrogenic responses.

Heterologous expression systems have been used to purify and characterize several proteins, including steroid hormone receptors (Metzger et al., 1988Go; Wooge, 1992). Many fusion proteins exhibit activity comparable to that of their native forms (Jaglaguier et al., 1996Go; Wittliff et al., 1990Go). Expressing proteins as fusions facilitates the production of significant quantities of the desired regions or mutations of interest and their purification. In addition, it allows for precise control of assay conditions (protein concentration, metabolism, and background proteins) making direct comparisons among different species possible. The affinity of the bacterially expressed GST-ERdef fusion proteins for E2 was in agreement with the Kd values reported for full length ERs from human and other species (Nimrod and Benson, 1997Go; Pakdel et al., 1990Go; Vonier et al., 1997Go; Wooge, 1992). However, the affinity of the GST-rtERdef for E2 was approximately 10-fold higher than that reported for full length rainbow trout ER (Le Drean et al., 1995Go; Pakdel et al., 1990Go). This discrepancy may be attributed to differences in protein purity, assay conditions and the lack of accessory proteins or differences in post-translational modifications. In addition, a wide range of Kd values have been reported for some species, for example the Kd determined from Xenopus liver cytosol ER has been reported to vary from 0.5 to 15 nM (Lutz and Kloas, 1999Go; Westley, 1978). This suggests that differences in protein preparation and assay conditions may also contribute to the variability in the reported Kd values.

It has been demonstrated that the degree of chlorination and the substitution pattern of PCB congeners can significantly influence their estrogenic properties (Korach et al., 1988Go; Moore et al., 1997Go). X-ray crystallography studies have demonstrated that ortho-substitution causes severe conformational restriction about the inter-ring bond, and conformationally restricted hydroxylated PCBs have been shown to be effective ligands for the ER (Korach et al., 1988Go). Quantitative structure activity relationships (QSARs) have also suggested that PCBs containing ortho- and para-chlorinated substituents are capable of binding to the ER (McKinney and Waller, 1994Go; Waller et al., 1995Go). In this study, the only PCBs found to interact with GST-hER{alpha}def and GST-aERdef receptors were three tetra-ortho-substituted PCBs, a penta-chlorinated PCB (PCB 104), and two hepta-chlorinated PCB (PCB 184, and PCB 188) congeners. This is in agreement with reports showing that PCB 104 was able to compete with [3H]E2 for binding to mouse uterine ER and induce ER-mediated gene expression (Fielden et al., 1997Go) and that PCB 188 and 104 induce MCF-7 cell proliferation (Andersson et al., 1999Go). PCB 54, the fourth tetra-ortho substituted PCB examined, exhibited a weak interaction with the GST-aERdef protein and did not bind to the GST-hER{alpha}def, which agrees with results reported by Arcaro et al. (1999) using a recombinant hER{alpha} preparation. Conversely, several PCB congeners, including PCB 104, 184, and 188, bound to the GST-rtERdef fusion protein, with the degree of interaction increasing as the number of chlorinated substituents increased. Many of the congeners that displaced at least 50% [3H]E2 from the GST-rtERdef fusion protein also contained at least one para-chlorinated substituent in addition to the ortho substitutions. Of the environmentally relevant PCBs, only PCB 45, 47, 91, and 177 competitively displaced at least 50% [3H]E2 from the GST-rtERdef fusion protein, however none of these congeners bound to the GST-hER{alpha}def or GST-aERdef fusion proteins.

The differences in PCB interaction between the GST-hER{alpha}def, GST-aERdef and GST-rtERdef fusion proteins may be due to amino acid-sequence differences among the receptors, particularly, the amino acids that form the binding pocket. Indeed, the promiscuity of the ER has been partially attributed to the size of the ligand-binding pocket, which is approximately 2 times the volume of E2 (Brzozowski et al., 1997Go). Amino acid sequence alignments of the ERs from different species reveal that the region of the receptor involved in ligand binding is variable. For example, the LBD of the hER{alpha} shares 90% amino acid sequence identity with the mouse ER{alpha}, 82% with the chicken ER, 79% with the green anole ER, 70% with the xenopus ER, and only 40% with the rainbow trout ER. However, identification of critical amino acid residues or motifs within the LBDs that contribute to observed differences in ligand preference and relative binding affinity through simple amino acid sequence alignment may be difficult. Despite differences in sequence identity of these ERs, the sequences from all species harbor the same 3 equivalent amino acid residues, Glu 353, Arg 394, and His 524, which participate in direct hydrogen bonds with E2 to stabilize the agonist in the binding pocket (Brzozowski et al., 1997Go).

Rodents treated with Aroclors experience a variety of estrogenic responses, including increases in uterine glycogen content and uterine wet weight (Ecobichon and MacKenzie, 1974Go). However, there have been few studies examining the ER binding affinities of these mixtures. Aroclor 1221 and 1254 have been shown to weakly bind the rat uterine ER (Nelson, 1974Go) while Aroclors 1221, 1248, and 1268 are capable of displacing [3H]E2 from the rainbow trout ER expressed in yeast (Petit et al., 1997Go). Aroclors 1221 and 1248 (10 and 100 µM, respectively) have also been reported to induce vitellogenin synthesis in rainbow trout hepatocytes (Petit et al., 1997Go). However, none of the Aroclors examined in this study were found to bind to any of the GST-ERdef fusion proteins. Complete congener analysis of 8 Aroclor mixtures (1221, 1232, 1242, 1016, 1248, 1254, 1260, and 1268), using capillary gas chromatography, demonstrated that PCBs 104, 184, and 188 are not detectable or are present at concentrations less than 0.05% wt (Schulz et al., 1989Go). In addition, none of the PCB congeners found to preferentially bind to GST-rtERdef were observed to exceed 2.6% wt (Schulz et al., 1989Go), resulting in a concentration that is unable to bind to the ER. The discrepancies between our results and those reported in the literature may be due to different assay conditions, measured endpoints, and differences in metabolic activity within the assays. It is well known that hydroxylation of select PCB congeners significantly increases their affinity for the ER (Fielden et al., 1997Go; Korach et al., 1988Go), thus suggesting that hydroxylation of PCB congeners plays an important role in the in vivo estrogenicity of Aroclor mixtures.

Although, the major HO-PCBs identified in human serum (HO-PCB 1–7) examined in this assay have been shown to significantly inhibit ER-mediated gene expression in transiently transfected MCF-7 cells (Moore et al., 1997Go), none of the congeners were found to compete with [3H]E2 for binding to the GST-ERdef fusion proteins. These results are similar to those reported by Kuiper et al. (1998) using baculovirus expressed hER{alpha} and rat ERß preparations. HO-PCB 7 is a para-hydroxylated metabolite of PCB 187; however, the hydroxylation of this congener did not increase its affinity for any of the GST-ERdef fusion proteins. This was in contrast to the para-hydroxylation of PCB 54 and 104, which significantly increased the affinity of the HO-PCBs for the ER of all 3 species. HO-PCB X, which binds both mouse and rat uterine ER, also bound to all 3 GST-ERdef fusion proteins. Unlike the fully ortho-chloro-substituted HO-PCB congener HO-PCB 54 (2,6,2',6'-tetrachloro-4-biphenylol), HO-PCB X (2',3',4',5'-tetrachloro-4-biphenylol) contains a single ortho substitution, but was found to bind to all 3 GST-ERdef fusion proteins with a slightly lower affinity than HO-PCB 54. In addition, nonphenolic chloro-substituted HO-PCBs have been shown to effectively compete for binding to the ER (Kuiper et al., 1998Go), although HO-PCB 54 consists of both phenolic and nonphenolic chloro-substitutions and competes for binding to all 3 fusion proteins. This suggests that in addition to the degree of ortho substitution, the chlorination pattern and position of the hydroxyl are important determinants of ER binding as previously described (Connor et al., 1997Go; Korach et al., 1988Go).

Increasing DMSO concentrations to 10% was found to effect ligand preference and relative binding affinity of PCBs. In contrast, it had little effect on E2 interactions with GST-ERdef fusion proteins, suggesting that DMSO may increase the solubility of PCB congeners and their availability for receptor interaction. For example, PCBs that bound weakly to the GST-ERdef fusion proteins with 4% DMSO in the assay mixture exhibited a significant increase in binding affinity in solutions containing up to 20% DMSO (Fig. 7AGo). However, at a final concentration of 20% DMSO, a significant decrease in total binding was observed, indicating direct effects on protein function. This observation has important implications for assessment of relative ligand-binding affinities for the ER, since organic solvent concentration may markedly influence the binding of some substances.

These results demonstrate that ERs from different species exhibit differential ligand preferences and relative binding affinities for PCBs, which can be dramatically affected by solvent concentration. Although many of the environmentally relevant PCBs did not effectively compete with [3H]E2 for binding to the GST-ERdef fusion proteins, the data generated from this study can be used for further development of ER QSARs (Waller et al., 1995Go) and also help in the derivation of species-specific QSARs (Tong et al., 1997Go).

In summary, we report the cloning of the first complete reptilian ER DEF sequence, which has been used in a study comparing the differential binding of PCBs and HO-PCBs to the ERs from human, green anole, and rainbow trout using a semi-high throughput, competitive binding assay. Surprisingly, several examples of differences in the absolute and relative binding affinity of a number of structurally-related PCBs among the GST-hER{alpha}def, GST-aERdef, and GST-rtERdef proteins were observed. The lack of differences between binding affinities for the human and green anole proteins is most likely due to the higher degree of amino acid sequence identity throughout their ligand binding domains. The most notable differences were observed between the GST-rtERdef and either of the other two GST-ER fusion proteins. This may have implications for risk assessment when extrapolating data between two such divergent species as humans and rainbow trout. Studies are currently underway that examine more structurally diverse substances, including pharmaceuticals, natural products, environmental pollutants, and industrial chemicals, for potential differences in ER-binding affinity across species.


    ACKNOWLEDGMENTS
 
We thank Stephen Safe for providing several of the PCB congeners, HO-PCB, and the commercial Aroclor mixtures. We also wish to thank Matts Tysklind and Patrik Andersson for their helpful comments and for providing some of the PCBs used in this study, and Juli Wade for providing the green anole liver tissue. The authors are also grateful to Stephen Safe, Trine Celius, Rob Halgren, Kirsten Fertuck, and Mark Fielden for their critical reading of this manuscript. Funding for this research was provided by the United States Environmental Protection Agency (R 826301–01–0) and the Chemical Manufacturers Association.


    NOTES
 
1 To whom correspondence should be addressed at Michigan State University, Department of Biochemistry, 419 Biochemistry Building, Wilson Road, East Lansing, Michigan, 48824–1319. Fax: (517) 353-9334. E-mail: tzachare{at}pilot.msu.edu. Web site: http://www.bch.msu.edu/~zacharet/tzlab.htm. Back


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