* Institute for Risk Assessment Sciences (IRAS), Utrecht University, Utrecht, NL-3508 TD The Netherlands;
Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, 3584 CT The Netherlands;
Institute for Environmental Studies, Vrije Universiteit, Amsterdam, NL-1081 HV The Netherlands;
Department of Environmental Chemistry, Stockholm University, Stockholm, S-101 06 Sweden; and
¶ Department of Zoology, National Food Safety and Toxicology Center, Institute of Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824
Received April 10, 2002; accepted June 18, 2002
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ABSTRACT |
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Key Words: polychlorinated biphenyls; methylsulfone metabolites; estrogen-responsive cells; in vitro bioassays; antiestrogenicity; gene expression.
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INTRODUCTION |
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A growing number of xenobiotics that possess antiestrogenic activities have been identified, i.e., compounds that antagonize estrogen-dependent processes in target tissues. Antiestrogenic substances may interfere at several sites with estrogen-regulated cellular processes, both by estrogen receptor (ER)-dependent ( or ß) and -independent mechanisms (Navas et al., 1998
). Major examples of synthetic antiestrogens that are active via ER-independent and AhR-dependent mechanisms are polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), and dioxin-like non- and mono-ortho PCBs (Gierthy et al., 1993
; Jansen et al., 1993
; Safe, 1994
). Tamoxifen was specifically designed to function via competitive interaction with the ER, and is used clinically as an antiestrogenic drug in the treatment of estrogen-dependent human breast cancer (Webb et al., 1995
).
Recent studies have reported that the endocrine-related activities associated with PCBs are mediated, in part, through the formation of hydroxylated (OH) and methylsulfonyl (MeSO2) PCB metabolites. OH-PCB metabolites possessing an OH group mainly in the para position, and chlorinated on the OH-containing phenyl ring have been identified almost exclusively in the blood of wildlife and humans (Bergman et al., 1994; Letcher et al., 2000a
; Sandau et al., 2000
). The estrogenic activity of retained, and thus environmentally relevant, OH-PCB congeners via interaction with the ER appears not to be very significant. Retained OH-PCBs have been shown in vitro to weakly bind to ER
or ERß (Kuiper et al., 1998
), and weakly induce gene expression in estrogen-responsive cell systems (Kramer et al., 1997
; Matthews and Zacharewski, 2000
; Moore et al., 1997
).
PCBs with 2,5-dichloro- or 2,5,6-trichloro-substitution, and thus meta-para hydrogens on at least one phenyl ring are biotransformed into persistent and bioaccumulative 3- and 4-MeSO2-PCB metabolites (Bakke et al., 1999). With respect to endocrine disruption-related effects, persistent MeSO2-PCBs are known to cause chronic and reproductive toxicity in exposed mink (Lund et al., 1999), bind to mouse and human glucocorticoid receptors (Johansson et al., 1998b
), are cytotoxic to human placental JEG-3 and JAR choriocarcinoma cells (Letcher et al., 1999
), are thyroidogenic in rats (Kato et al., 1999
), competitively inhibit CYP11B1 activity in mouse Y1 adenocortical cells (Johansson et al., 1998a
), and inhibit CYP11ß activity in the adrenal of Baltic grey seal (Lund, 1994
). In humans and some wildlife species 2,2-(4-chlorophenyl)-1,1-dichloroethene (4,4-DDE), a metabolite of the insecticide 2,2-(4-chlorophenyl)-1,1,1-trichloroethane (4,4-DDT) is biotransformed to a persistent 3-MeSO2-4,4-DDE metabolite (Letcher et al., 2000a
,b
; Weistrand and Norén, 1997
). To our knowledge, no previous studies have been conducted of ER-binding affinities or antiestrogenic activity in vitro or in vivo via agonism or antagonism of estrogen-responsive gene expression by MeSO2-PCBs or 3-MeSO2-4,4-DDE.
In vitro assays developed for the routine screening of potential xenoestrogens include proliferation of MCF-7 human breast cancer cells, competitive ER binding assays, and induction of reporter gene expression in transiently or stably infected cell lines (Ankley et al., 1998; Reel et al., 1996
). The present study investigated the estrogenic and antiestrogenic effects of the highly persistent and bioaccumulative 3- and 4-MeSO2-CB49 and -CB101, and 3-MeSO2-4,4-DDE on estrogen-responsive gene expression in four in vitro assays. The effects of the precursors CB101 and 4,4-DDE, the MeSO2-PCB analog MeSO2-2,5-dichlorobenzene, and the known ER antagonists, tamoxifen and ICI 182,780 were also examined. Agonistic and antagonistic effects on 17ß-estradiol- (E2)-induced luciferase activity were determined in three different ER-CALUX (estrogen-responsive, chemically activated luciferase expression) assays; recombinant human breast ER-CALUX adenocarcinoma cells (Legler et al., 1999
), and recombinant human embryonic kidney HEK293-ER
-ERE.luc and HEK293-ERß-ERE.luc cells (Lemmen et al., submitted). Agonist/antagonist effects on E2-induced vitellogenin (vtg) production were assessed in an assay using hepatocytes from male carp fish (Cyprinus carpio) (Smeets et al., 1999
).
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MATERIALS AND METHODS |
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The three assays using stably transfected cell lines were performed as previously described for T47D cells (Legler et al., 1999). Cells were trypsinized, resuspended in assay media, and seeded in 96-well plates (Packard, Meriden, CT) at a density of 5000 cells per well in 100 ml of HEK293-ER
-ERE.luc, and HEK293-ERß-ERE.luc cells were seeded in 96-well plates (NUNC) at a density of 15,000 cells per well in 200 ml of assay medium. The assay media were steroid-free by using phenol red-free DF and fetal calf serum treated with 5% dextran-coated charcoal (DCC-FCS; Horwitz and McGuire, 1978
). After 24 h, the ER-CALUX cells were approximately 50% confluent, and the assay medium was renewed. HEK293-ER
-ERE.luc, and HEK293-ERß-ERE.luc cells were cultured without medium renewal for 48 h, after which time 5060% confluency was reached.
For the cells of the ER-CALUX assays, the assay medium was replaced by incubation medium 48 h after initial cell plating. Prior to the cell incubations with the compounds, the DMSO stock solutions were diluted 1000-fold in assay medium. The nominal concentration of the compounds in the assay medium ranged from 0.05 to 20 µM. For each set of experimental plates a complete E2 standard curve (1 to 100 pM) was included. In addition, The EC50 concentration (10 pM) of E2 was included in every 96-well plate. For all three ER-CALUX assays, all compound concentrations were tested in two sets of three replicates. DMSO concentrations in the assay medium did not exceed 0.2% (v/v).
After an incubation period of 24 h at 37°C in an atmosphere of 7.5% CO2, ER-CALUX plates were transferred to ice, and the assay medium was removed. To each well, 50 ml of lysis buffer was added, containing 1% (v/v) Triton X-100, 25 mM glycylglycine, 15 mM MgSO4, 4 mM EDTA (pH 7.8), and 1 mM DTT. After vigorous shaking of the plates at 4°C for at least 10 min, 25 ml of the lysate suspension was transferred to a 96-well view plate (Packard). To each well, 25 ml of LucLite solution was added. The Luclite luciferase reporter gene assay system (Packard BioScience B.V., Groningen, The Netherlands) was used to measure luciferase activity, according to the manufacturers instructions. Alternatively, cells were washed twice with 100 ml of phosphate-buffered saline (PBS), and subsequently lysed in 30 ml low-salt (LS) buffer containing 10 mM Tris (pH 7.8), 2 mM dithiotreitol (DTT), and 2 mM 1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid. Following a 10-min incubation on ice, the 96-well plates were frozen at 80°C for a minimum of 30 min and maximum of 1 day to lyse the cells. The plates were thawed on ice and shaken for 5 min at room temperature. Luciferase activity was measured in a luminometer (Labsystems Luminoscan RS) with automatic injection of 100 ml flash mix (pH 7.8) per well containing 470 mM luciferin, 20 mM trycine, 1.07 mM (MgCO3)4Mg(OH)2.5H2O, 2.67 mM MgSO4, 0.1 mM EDTA, 5 mM ATP, and 2 mM DTT (pH 7.8).
CARP-HEP/vtg assay.
The common carp (Cyprinus carpio) used in the carp hepatocyte/vitellogenin (CARP-HEP/vtg) assay were genetically uniform, all male (XY), F1 hybrid progenies. Further details of the carp, the maintenance of the fish prior to use in the assay, and the perfusion procedure are described in detail in Smeets et al.(1999). Briefly, carp hepatocytes were freshly perfused by a 2-step retrograde technique, isolated, and cultured. The liver was first perfused with Ca2+- and Mg2+-free buffer containing EDTA (0.145 M NaCl; 5.4 mM KCl; 5 mM EDTA; 1.1 mM KH2PO4; 12 mM NaHCO3; 3 mM NaH2PO4; 100 mM HEPES; pH: 7.5) and followed by a step with the same buffer containing no EDTA and 0.26 mg/ml collagenase D (Boehringer, Mannheim, Germany). The perfused liver sections were filtered through nylon mesh. After removal, mincing, sieving, and washing, the hepatocytes were resuspended in culture medium. The cell viability was >90% as assessed with trypan-blue staining.
The isolated hepatocytes were cultured in phenol red-free DMEM/F12 medium (D2906, Sigma Chemical Co., St. Louis, MO) supplemented with 14.3 mM NaHCO3, HEPES (final concentration: 20 µM), 50 mg/l gentamycin, 1 µM insulin, 10 µM hydrocortisone, 2% v/v Ultroser SF (steroid-free) serum (Jones Chromatography, Mid Glamorgan, UK), and 2 mg/l of the protease-inhibitor aprotinin (Fluka, Buchs, Switzerland) at pH 7.4. The concentration of the cell suspension was 1.0 x 106 cells/ml, and 0.18 ml (or 180,000 cells) was added to each well of 96-well tissue plates (Greiner, Alphen a/d Rijn, the Netherlands). The plates were maintained at 24°C for a period of 36 h to acclimatize the cells. The proportion of erythrocytes relative to hepatocytes did not exceed 10%.
After the 36-h acclimatization period, the cell confluence was about 70% and was ready for dosing. All DMSO stock solutions of compounds and concentrations used in the ER-CALUX were diluted 1000-fold in assay medium and used in the CARP-HEP/vtg assay. The culture medium (90%) was replaced by assay medium (164 µM) containing 10% higher compound concentrations to obtain the desired 1000-fold dilution. After two days, the assay medium (90%) was refreshed with new assay medium containing nominal compound and E2 concentrations. After a further twodays, the medium was transferred to new 96-well plates, and frozen at 70°C until further use. The remaining hepatocytes were used to determine EROD activity, cell viability, or protein content. An indirect competitive ELISA was used to quantify the vtg present in the assay medium. The ELISA procedure, as well as calculations to quantify vtg, have been thoroughly described (Smeets et al., 1999). For each experiment set, an E2 dose-response curve plate was included with concentrations ranging from 0.6 to 6000 nM. An E2 positive control (EC50, 100 nM) was included on all other plates. All E2 and compound concentrations were tested in two separate sets of 6-fold replicates. The DMSO concentration in the assay medium did not exceed 0.2% (v/v).
Antiestrogenic effects.
Antiestrogenic activity of the test compounds was assessed in the in vitro assays using the same nominal concentrations, procedures, and number of replicates used in the estrogenicity screening. The ER-CALUX, HEK293-ER-ERE.luc and HEK293-ERß-ERE.luc cells were co-administered with 10 pM (EC50) or 100 pM E2 in separate co-incubations with the different test compounds and concentrations. In the CARP-HEP/vtg assays, the hepatocytes were co-administered with 100 nM (EC50) or 20 nM E2. For both the CALUX and CARP-HEP/vtg assays, E2 concentrations differing from the EC50 by 10 times and 5 times, respectively, were used to assess the competitive antagonism of the test compounds with E2-induced gene expression. An E2 amount 5 times lower than the EC50 concentration, rather than 10 times higher as in the CALUX assays, was used in the CARP-HEP/vtg assays because 3-MeSO2-CB49 antagonized E2-induced vtg production at 20 nM (Fig. 2A
), but not at 100 nM (Fig. 2B
). If 1000 nM E2 (10 times the EC50) had been used, no competitive reduction in the 3-MeSO2-CB49 antagonism would have been observed. DMSO present in the compound incubations did not exceed 0.2% (v/v). In all four in vitro assays, 0.1, 1.0, and 10 µM of the known competitive ER antagonist tamoxifen were included as positive controls for antiestrogenicity. In addition, 0.25, 2.5, and 10 nM concentrations of the ER antagonist ICI 182,780 were also included as positive controls in the ER-CALUX and 293-ER
-ERE assays. Tamoxifen and ICI 182,780 inhibited >90% of the E2-induced (10 pM) gene expression at 10 µM and 10 nM, respectively.
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Cell viability.
A possible cytotoxic effect evoked by the compounds in the ER-CALUX cells and carp hepatocytes was assessed by changes in the mitochondrial dehydrogenase-mediated metabolism of 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT), or the MTT test (Denizot and Lang, 1986). The procedure for the ER-CALUX cells were similar to Meerts et al.(2001). After vtg harvesting, the remaining hepatocytes were incubated with MTT for 1 h, similar to Smeets et al. (1999) with minor modifications.
AhR-CALUX assay, EROD activity in carp hepatocytes and protein content.
The MeSO2-PCBs, or more likely, dioxin-like impurities in the pure compound samples, were assessed for AhR-mediated activity. After vtg harvesting, carp hepatocytes were frozen or used immediately for the determination of CYP1A enzyme activity by measurement of ethoxyresorufin-O-deethylase (EROD) activity (Burke and Mayer, 1974; Smeets et al., 1999
). A recombinant H4IIE rat hepatoma (H4IIE.luc) cell line, or AhR-CALUX, containing a luciferase reporter gene under the control of dioxin-responsive enhancers, also was used to assess the possible dioxin-like activity and was performed as described elsewhere (Sanderson et al., 1996
). TCDD concentrations of 0.3, 1.0, 10, 100 and 1000 pM were measured as positive controls.
Protein content was determined for dosed and control carp hepatocytes according to the procedure of Lowry et al.(1951) and Rutten et al.(1987), with some modifications. After vtg harvesting, the hepatocytes were washed twice with 200 ml of phosphate-buffered saline (PBS). An aqueous solution of 0.5% Triton X-100 (v/v, 200 ml) was then added. The plates were frozen at 70°C for at least 2 h. After thawing to room temperature, the protein content in each well was diluted, if necessary, for protein determination by comparison relative to a BSA standard curve.
Dose-response curves, calculations and statistics.
Dose-response relations were described by the sigmoidal function y = a0 + a1/(1 + exp([a2 + x)/a3]) (SlideWrite Plus, 4.0, Advanced Graphics Software, Carlsbad, CA), in which y is the induction of or remaining (%RR, equation 1) luciferase activity or vtg production, and x is the logarithm of the dose. For estrogenic effects, vtg or luciferase levels were normalized to the maximum levels induced by E2, and expressed as a percent of the maximum level. For antiestrogenic effects, the levels were normalized to the vtg or luciferase induced by the co-administered E2 concentration alone. The statistical significance of the differences between compound-treated and control cells were determined using a 2-way ANOVA (p < 0.05).
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RESULTS |
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The LOEC (4.1 nM) and EC50 (100 nM) concentrations of E2 for vtg production in the CARP-HEP assay were similar to the findings by Smeets et al.(1999). These potencies were 3 to 4 orders of magnitude lower than in the three ER-CALUX assays. Variability in the sensitivity to E2 among carp hepatocytes cultures was not observed in our study, which was a similar finding to E2-induced vtg production in hepatocytes from the same genetically uniform strain of male carp used in Smeets et al.(1999). However, the efficacy (>1000-fold) of vtg production was 2 orders of magnitude higher than that described in Smeets et al.(1999). Pelissero et al. (1993) reported variability in E2 sensitivity for vtg expression among different cultures for rainbow trout (Oncorhynchus mykiss) hepatocytes. Pelissero et al.(1993) suggested that, considering the ER itself is induced by exposure to E2, ER and vtg gene transcription, mRNA stability for vtg, ER turnover, E2 metabolism in culture, and the rate of exocytosis of vtg, all factor into the rate of vtg secretion into the surrounding medium. We also recently demonstrated that exposure of carp hepatocytes to low or background levels of estrogens and xenoestrogens (e.g., methoxychlor and bisphenol A) can increase the sensitivity for vtg production from exposure to endogenous estrogens (Rankouhi et al., 2002
). Therefore, it is necessary to compare xenoestrogen responses to an E2 dose-response curve obtained in the same hepatocytes culture, as was done in the present study.
E2-induced vtg production (EC50) was inhibited essentially 100% at tamoxifen concentrations approaching 10 µM. Using the CARP-HEP/vtg assay, the LOEC and IC50 concentrations (Table 1) were calculated from dose-response curves with 100 nM E2 (EC50) and 0.01, 0.03, 0.1, 0.3, 0.7, 1.0, 3.0, or 10 µM of tamoxifen (not shown). For the ER-CALUX the LOEC and IC50 concentrations (Table 1
) were with 10 pM E2 (EC50) and 0.00005, 0.0005, 0.01, 0.1, 0.5, 1.0, or 10 µM of tamoxifen (not shown). The IC50 and LOEC concentrations were 1 and 3 orders of magnitude greater, respectively, than for tamoxifen inhibition of luciferase activity in the T47D.luc cells (Table 1
). Tamoxifen alone did not induce vtg production up to 10 µM.
In vitro antiestrogenic activity of the aryl methyl sulfones and their precursors.
The MeSO2-PCBs, CB101 and MeSO2-2,5-Cl2Bz (Fig. 1) were examined for agonist and antagonist activity in the four estrogen-responsive in vitro assays. MeSO2-2,5-Cl2Bz has the same functional group substitution as the MeSO2-containing phenyl ring of the 4-MeSO2-PCBs (Fig. 1
). This permitted an evaluation of the biphenyl backbone in any observed estrogenic or antiestrogenic activity of the MeSO2-PCBs. 4,4-DDE (10 µM)-induced luciferase activity about 17-fold relative to the DMSO control, whereas E2-induced activity was not significantly (p < 0.05) antagonized (103 ± 11% of EC50). 3-MeSO2-4,4-DDE (10 µM) did not significantly (p < 0.05) induce luciferase activity (1.3-fold relative to control) or antagonize (88 ± 9% of EC50) E2-induced luciferase activity. 4,4-DDE and 3-MeSO2-4,4-DDE were not screened in the remaining assays.
The 3- and 4-MeSO2-CB49 and -CB101 congeners, CB101 and MeSO2-2,5-Cl2Bz did not significantly (p < 0.05) induce ER-mediated gene expression in any of the four in vitro assays up to maximum concentrations of 10 or 20 µM. Concentrations up to 10 µM of CB101 and MeSO2-2,5-Cl2Bz, and in the presence of an EC50 concentration of E2, did not significantly (p < 0.05) antagonize E2-induced responses in the assays.
With the exception of 3-MeSO2-CB49, the MeSO2-PCBs consistently antagonized the E2-induced response (EC50 concentration) in a concentration-dependent manner in all four in vitro assays (Table 1, Figs. 2 to 5
). Based on LOECs and IC50 concentrations (Table 1
), the antiestrogenic effect was in the order of 4-MeSO2-CB101 > 4-MeSO2-CB49 > 3-MeSO2-CB101 >> 3-MeSO2-CB49. Thus, the 4-MeSO2-PCBs were generally more effective anti-estrogens relative to the 3-MeSO2-PCB congeners. The E2-induced response (EC50) in the presence of 4-MeSO2-CB101 (41 ± 7%) and -CB49 (64 ± 4%) reached an apparent maximum in the CARP-HEP/vtg assay (Fig. 2B
). IC50 concentrations were about 5.0 µM. In the ER-CALUX assay, the remaining E2-induced activity (EC50) for 4-MeSO2-CB101 (28 ± 4%) and -CB49 (39 ± 3%) were lower, but did not appear minimized at the maximum treatment concentration of 10 µM (Fig. 3A
). In the ER-CALUX cells, the estimated IC50 concentrations for 4-MeSO2-CB101 and -CB49 were about 2 orders of magnitude lower when compared to the CARP-HEP/vtg assay (Table 1
).
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Mechanism of the MeSO2-PCB-mediated, antiestrogenic effect.
In the ER-CALUX assay, the antagonistic effect of 3- and 4-MeSO2-CB101 and 4-MeSO2-CB49 were largely reduced by exposure to E2 at 10 times (100 pM) the EC50 concentration (Fig. 3B). In the CARP-HEP/vtg assay, an E2 concentration 5 times lower (20 nM) than the EC50 facilitated an increase in the inhibitory effects of 4-MeSO2-CB101 and -CB49 (Fig. 2A
). Antagonistic effects on E2-induced activity were observed for 3-MeSO2-CB49 at concentrations of 10 and 20 µM, where >60% of the E2-induced activity was maintained.
In the ER-CALUX and CARP-HEP/vtg assays, for any compound at any of the concentrations tested (up to 10 µM), the MeSO2-PCBs, tamoxifen or ICI 182,780 did not influence cell viability as indicated by a lack of significant (p < 0.05) change in the MTT activity relative to the DMSO or blank controls. AhR-induced effects of the TCDD controls were observed on E2-induced (EC50) vtg production and EROD activity in the CARP-HEP assay, whereas only 3-MeSO2-CB101 (at 10 µM) demonstrated any effect (Table 2). In the presence of E2 (EC50), 10 and 100 pM TCDD inhibited the production of vtg by 13 ± 5% and 64 ± 6%, respectively (not shown). With the exception of 3-MeSO2-CB101, the MeSO2-PCBs were not AhR-active up to 10 µM in the AhR-CALUX assay (Table 2
). The same TCDD concentrations as used for CARP-HEP/EROD activity inhibited E2-induced luciferase activity (not shown), similar to previous studies (Sanderson et al., 1996
).
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DISCUSSION |
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The present study demonstrated that persistent 3- and 4-MeSO2-CB49 and -CB101 metabolites do not agonize transactivation of estrogen-responsive reporter genes in in vitro assays based on recombinant human cells or primary carp hepatocytes. However, MeSO2-PCBs, especially 4-MeSO2-PCBs, are capable of inhibiting estrogen-responsive gene expression in a concentration- and structure-dependent manner by interaction with human or fish ER. Higher E2 concentrations partially reversed the inhibitory effects of the MeSO2-PCBs in all four assay systems, which demonstrates that the antagonist interaction with substrate binding site on the ER is at least reversible (or surmountable).
MeSO2-PCB antiestrogenicity was dependent on the presence and position of the MeSO2-functional group and the chlorinated (substitution pattern) biphenyl backbone. 4-MeSO2-substitution, rather than 3-MeSO2-substitution, resulted in more effective MeSO2-PCB antiestrogenicity. CB101 is the non-MeSO2-containing base structure of 3- and 4-MeSO2-CB101, yet the parent compound itself was not estrogenic or antiestrogenic. Thus, our study shows that the intact MeSO2-PCB structure appears to be essential for antagonistic interaction with both the carp and human ERs, and is optimized by 4-MeSO2-substitution. The increased inhibitive effect of increasing MeSO2-PCB concentrations in competition with E2 concentrations, similar to tamoxifen, further exemplified that the antagonism of E2-induced activity proceeds, most likely, via direct interaction with the human and carp ERs. The structural diversity of potential ER antagonists is also shown by tamoxifen, which lacks the phenolic A-ring of E2 (Fig. 1), yet competitively binds to the ER. Tamoxifen can be agonistic as well as antagonistic, depending on the target tissue and the species (Nimrod et al., 1997
).
Non-ER-mediated mechanisms are an unlikely explanation of the observed MeSO2-PCB antiestrogenic effects. The antiestrogenicity of MeSO2-PCBs was not mediated via concentration-dependent cytotoxic effects, as shown by the negative effects on MTT activity. CYP1A1/1B1 activity has been reported in wild-type T47D and the ER-CALUX cells derived from the T47D cells (Legler et al., 1999; Spink et al., 1998
), and carp hepatocytes possess inducible CYP1A1 activity (Smeets et al., 1999
). However, in situ transformation of MeSO2-PCBs to active, antiestrogenic metabolites was unlikely in these cell systems because 3- and 4-MeSO2-CB101 have no adjacent, hydrogen-bound carbons on the biphenyl structure, which is a necessary substrate feature for Phase I, CYP enzyme-mediated PCB biotransformation (McFarland and Clarke, 1989
). The hydrogen substitution patterns of 3- and 4-MeSO2-CB101 are analogous to 2,2,4,4,5,5-hexachlorobiphenyl (CB153), which has a biological half-life in humans estimated to be 338 days (Bühler et al., 1988
). Furthermore, in species where MeSO2-PCB metabolites have been shown to be formed, 3- and 4-MeSO2-CB49 and CB101 are increasingly dominant residues relative to other MeSO2-PCB congeners, which indicates the greater resistance of these metabolite structures to further metabolism (Letcher et al., 2000a
Letcher et al., 1998).
Compounds may interfere with E2-regulated cellular processes by ER-dependent mechanisms, but also ER-independent mechanisms, so as to decrease the effectiveness of the E2-mediated response (Navas et al., 1998). For example, AhR-mediated induction of CYP1A enzymes can evoke dose-dependent hydroxylation of E2, and thus decrease the level of E2 that is present, and therefore bioavailable to the cell. The antiestrogenic activity of AhR agonists such as TCDD and related dioxins and furans, PCBs, and PAHs in cell systems is often a direct result of their binding affinity for the AhR and the subsequent induction of CYP1A activity (Hoivik et al., 1998
). TCDD and non-ortho (co-planar) PCBs are antiestrogens that can also function via an AhR-mediated mechanism of action involving "cross talk" between ER and AhR signaling pathways (Safe et al., 1998
). These AhR-dependent pathways are improbable mechanisms for the observed MeSO2-PCB antiestrogenic activity in our bioassay systems since interaction with the AhR and subsequently mediated activities was minimal if existing at all.
Our findings indicate that an AhR-mediated mechanism similar, e.g., to TCDD (Safe et al., 1998) is not an explanation for the observed MeSO2-PCB antiestrogenicity. With the exception of 3-MeSO2-CB101, the MeSO2-PCBs were not AhR-active in the AhR-CALUX and CARP-HEP/EROD assays. Direct AhR-interaction by 3-MeSO2-CB101 is unlikely since persistent MeSO2-PCBs are not expected to have affinity for the AhR. 3- and 4-MeSO2-CB49 and CB101 contain 2 ortho chlorines. By analogy to di-ortho PCBs, persistent MeSO2-PCBs cannot attain the obligatory co-planarity of the phenyl rings necessary for a dioxin-like structure (Safe, 1994
) and thus are expected to have a low binding affinity for the AhR. In a human lymphoblastoid cell line (Kiyohara et al., 1990
), 10 of the 11 MeSO2-PCB congeners tested (at about 2 µM) did not induce CYP1A-mediated, benzo[a]pyrene hydroxylase (AHH) activity. The AhR-mediated EROD and luciferase activity of 3-MeSO2-CB101 in this study can perhaps be explained by the presence of a dioxin-like impurity formed during the chemical preparation of the compound. Assay treatment levels of MeSO2-PCBs were in the µM range, and therefore a pM level amount of a dioxin-like impurity would represent less than 0.1% of the amount of MeSO2-PCB present in the standard used.
The antiestrogenicity of persistent MeSO2-PCB towards E2-stimulated gene expression in vitro appears to be more significant than for environmentally relevant OH-PCB metabolites. Whereas the 4-MeSO2-CB49 and CB101 were effective inhibitors of E2-responsive gene expression in the present assay systems, environmentally relevant OH-PCBs have shown, i) little affinity for either the or ß subtype ER in humans, fish or amphibians, and ii) are weakly inhibitive of E2-induced gene expression in vitro (Kuiper et al., 1998
; Matthews et al., 2000).
The physiological significance of the inhibitive potency and efficacy observed for the MeSO2-PCBs is best compared to the competitive ER antagonist, tamoxifen. Tamoxifen (ICI 46,474; Nolvadex) is used clinically as a protective agent against estrogen-dependent breast cancer. The inhibitive LOEC potency for tamoxifen relative to 4-MeSO2-CB49 or -CB101 was only 100-fold and about 10-fold lower in the ER-CALUX and CARP-HEP/vtg assays, respectively. Human plasma concentrations of tamoxifen after a typical clinical dose is about 190 nM (Tukker et al., 1986), whereas concentrations of 4-MeSO2-CB49 and -CB101 have been reported to be about 0.02 nM in Swedish subjects (Weistrand et al., 1997
). Comparison of these data may suggest that the potential antiestrogenic impact to humans from MeSO2-PCB exposure is possibly many orders of magnitude lower then that of tamoxifen used in therapeutic doses. However, differences in metabolic susceptibility, and thus bioavailability, and bioaccumulative potential may suggest otherwise. Tamoxifen is given chronically to maintain steady-state levels in preventative treatment for breast cancers. After oral administration, the half-life of tamoxifen in human plasma was about 10 h, and is thus rapidly metabolized in vivo (Tukker et al., 1986
). Tamoxifen is also metabolized in vivo to a hydroxylated form, which is about 10 times more potent. The 4-MeSO2-CB49 and -CB101 concentrations of 0.02 nM reported in the plasma of Swedish subjects (Weistrand et al., 1997
) is about 5 orders of magnitude lower than the 1.0 µM LOEC found for antagonistic activity in the ER-CALUX assay (Table 1
). However, whether formed metabolically or transferred within food webs, the MeSO2-PCBs can accumulate in lipid-bearing target organs. For example, sum MeSO2-PCB concentrations in the Baltic grey seal have been recorded as high as 110,000 ng/g (lipid wt) in adipose tissue (Letcher et al., 2000a
), whereas levels ranging from 1.0 to 6.0 ng/g (lipid wt) have been reported in the plasma of Swedish human subjects (Weistrand et al., 1997
).
In HEK293 cells, MeSO2-PCBs antagonized E2-induced luciferase activity preferentially via an ER- versus a ERß-mediated mechanism of action. However, this is assuming that the two ER subtypes are expressed at equivalent levels in the two HEK293 cell lines, which was not verified in this study. Preference for ER
over ERß may also be true in the T47D.luc cells since they express both the
and ß forms of the ER subtypes, but were not differentiated in our experiments (Legler et al., 1999
). Differences in ligand structure can have profound effects on ER
versus ERß transactivational activity. Our observations of preferential interactions with human ER
for the tested MeSO2-PCBs are similar to findings with other xenobiotics. A number of estrogenic chemicals, including OH-PCBs and polycyclic musk compounds, preferentially transactivate luciferase expression in HEK293-ER
-ERE.luc relative to HEK293-ERß-ERE.luc cells (Kuiper et al., 1998
; Seinen et al., 1999
; Lemmen et al., submitted). Other examples include the efficacy and potency of a hydroxylated triaryl-pyrazole in comparison to E2, where the induction of CAT activity in ER
human endometrial cancer cells (ER
-HEC-1.CAT) were less than in ERß-HEC-1.CAT cells (Sun et al., 1999
). Sun et al.(1999) also showed that a structurally related tetrahydrocannabinol compound was also inductive in ER
-HEC-1.CAT cells, but was a full antagonist in ERß -HEC-1.CAT cells. The physiological significance of the higher affinity toward ER
relative to ERß is suggestive that MeSO2-PCBs differentially affect the expression of the two ER sub-types in humans (Kuiper et al., 1997).
In conclusion, the antagonism of E2-induced gene expression in vitro by persistent 3-, and especially 4-MeSO2-CB49 and -CB101, was concentration- and structure-dependent, regardless of the human or fish in vitro assay system used. The mechanism of 3- and 4-MeSO2-CB101 antiestrogenicity appears to be competitive antagonism of E2 interaction with the ER. However, further research, using multiple antagonist concentrations, on the complete E2 dose-response curve is necessary to unambiguously show that the MeSO2-PCBs are competitive antagonists. MeSO2-PCB interactions apparently favored ER over ERß in the human embryonic cell lines. Comparisons to tamoxifen indicated that especially 4-MeSO2-PCBs are relatively potent antiestrogens in these cell assays. The results of this study suggest that persistent and bioaccumulative MeSO2-PCB metabolites in exposed wildlife may be of concern as antiestrogens with the potential to modulate estrogen action at the level of the ER. It is also important to note that our findings with MeSO2-PCBs indicate that, in terms of environment contaminant exposures in wildlife and humans, antiestrogenicity might be equally important as estrogenicity with respect to impacts and risks to development and reproduction.
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ACKNOWLEDGMENTS |
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