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© 1999 Oxford University Press

Article

3,3',4,4'-Tetrachlorobiphenyl exhibits antiestrogenic and antitumorigenic activity in the rodent uterus and mammary cells and in human breast cancer cells

Kavita Ramamoorthy, Mona Sethi Gupta, Gulan Sun, Andrew McDougal and Stephen H. Safe1

Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843-4466, USA


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
3,3',4,4'-Tetrachlorobiphenyl (tetraCB) binds to the aryl hydrocarbon receptor (AhR), and several reports have demonstrated that AhR agonists exhibit antiestrogenic and antitumorigenic activities in human breast cancer cells, the rodent uterus and breast. In contrast, a recent study showed that 3,3',4,4'-tetraCB bound the estrogen receptor (ER) and exhibited ER agonist activities, and we therefore have reinvestigated the estrogenic and antiestrogenic activities of 3,3',4,4'-tetraCB. Our results showed that 3,3',4,4'-tetraCB and a structurally related analog, 3,3',4,4',5-pentaCB, did not bind the mouse uterine or human ER, did not induce proliferation of MCF-7 or T47D human breast cancer cells or induce reporter gene activity in cells transfected with E2-responsive constructs derived from the creatine kinase B (pCKB) or cathepsin D (pCD) gene promoters. Moreover, 3,3',4,4'-tetraCB and 3,3',4,4',5-pentaCB did not induce an increase in uterine wet weight, peroxidase activity or progesterone receptor binding in the 21–25-day-old female B6C3F1 mouse uterus. In contrast, both compounds inhibited 17ß-estradiol (E2)-induced cell proliferation and transactivation in MCF-7/T47D cells and uterine responses in B6C3F1 mice; surprisingly inhibition of E2-induced reporter gene activity was not observed in T47D cells transfected with pCKB, and this was observed as a cell-specific response with other AhR agonists. Additionally, 3,3',4,4'-tetraCB significantly inhibited mammary tumor growth in female Sprague–Dawley rats initiated with 7,12-dimethylbenzanthracene. Our results indicate that 3,3',4,4'-tetraCB does not exhibit ER agonist activity but exhibits a broad spectrum of antiestrogenic responses consistent with ligand-mediated AhR–ER crosstalk.

Abbreviations: AhR, aryl hydrocarbon receptor; BSA, bovine serum albumin; CAT, pBL/TATA/chloramphenicol acetyl transferase; DCC, dextran-coated charcoal; DMBA, dimethylbenzanthracene; DME/F-12, Dulbecco's modified Eagle's medium nutrient mixture F-12 Ham; E2, 17ß-estradiol; ER, estrogen receptor; ER{alpha}, estrogen receptor {alpha}; ERß, estrogen receptor ß; EROD, ethoxyresorufin O-deethylase; FBS, fetal bovine serum; hER, human estrogen receptor; MEM, minimum Eagle's medium; PAHs, polycyclic aromatic hydrocarbons; PBS, phosphate-buffered saline; PCB, polychlorinated biphenyl; pCD, cathepsin D; pCKB, creatine kinase B; PR, progesterone receptor; TCDD, tetrachlorodibenzo-p-dioxin; tetraCB, 3,3',4,4'-tetrachlorobiphenyl; UPO, uterine peroxidase assay.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Estrogen receptor {alpha} (ER{alpha}) and estrogen receptor ß (ERß) are ligand-activated transcription factors and members of the nuclear receptor superfamily (1–4). ER{alpha} and ERß bind estrogenic steroids such as 17ß-estradiol (E2) with high affinity and both ER subtypes bind structurally diverse compounds with similar but not identical affinities (5). Space-filling models of many synthetic ER ligands illustrate similarities in molecular size/shape and hydroxyl substitution that overlap with the 3- and 17-hydroxyl groups in E2 (6,7). Organochlorine compounds are among the most unusual ER ligands compared with the prototypical ligand E2; these compounds which include kepone, o,p'-DDT, dieldrin, and several polychlorinated biphenyl congeners do not contain hydroxyl substitution overlapping with the 3-hydroxyl moiety in E2 and their backbone structures are also different from E2 and other high affinity ligands for the ER (8–16). Several studies have reported that commercial polychlorinated biphenyl (PCB) mixtures and some congeners exhibit estrogenic activity (12–18); however, it is possible that some of these responses are due to hydroxylated metabolites which have been more extensively characterized as ER agonists (19–23).

Fielden et al. (12) recently confirmed that two ortho-substituted PCB congeners, 2,2',4,6,6'-penta- and 2,2',4,4',6,6'-hexachlorobiphenyl competitively bound to the mouse uterine ER and exhibited ER agonist activities in vitro and in vivo. 2,2',4',6,6'-Pentachloro-4-biphenylol, the expected metabolite of 2,2',4,6,6'-pentachlorobiphenyl, was also estrogenic; however, the in vitro estrogenic activities of the parent hydrocarbon in MCF-7 human breast cancer cells was observed in the absence of any hydroxy-PCB metabolite formation as determined by gas chromatographic–mass spectrometric analysis. Nesaretnam et al. (14) reported that 3,3',4,4'-tetrachlorobiphenyl (tetraCB) also bound the ER and exhibited estrogenic activity in in vitro bioassays. This result was surprising since previous studies have demonstrated that 3,3',4,4'-tetraCB inhibited E2-induced secretion of procathepsin D in MCF-7 cells (24) and similar results were observed for a series of structurally related halogenated aromatics which bound the aryl hydrocarbon receptor (AhR). These data were consistent with studies demonstrating that AhR agonists inhibit E2-induced responses in both in vivo and in vitro models via crosstalk between AhR and ER signaling pathways (reviewed in refs 25,26). The major objective of this study was to reexamine the estrogenic/antiestrogenic effects of 3,3',4,4'-tetraCB in ER binding assays, human breast cancer cells, the mouse uterus, and in the 7,12-dimethylbenzanthracene (DMBA)-induced mammary tumor model. Moreover, for some of these studies 3,3',4,4',5-pentachlorobiphenyl, a potent AhR agonist, was used as a positive control for AhR-mediated responses. The results showed that 3,3',4,4'-tetraCB alone was not an ER agonist but exhibited antiestrogenic activities and this was consistent with the AhR-mediated activity of this compound.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cells, chemicals and biochemicals
MCF-7 and T47D human breast cancer cells were purchased from the American Type Culture Collection (Rockville, MD). Dulbecco's modified Eagle's medium (DMEM) nutrient mixture F-12 Ham (DME/F-12) without phenol red, phosphate-buffered saline (PBS), acetyl CoA, E2 and 100X antibiotic/antimycotic solution were purchased from Sigma (St Louis, MO). Fetal bovine serum (FBS) was obtained from Intergen (Purchase, NY). Minimum Eagle's medium (MEM) was purchased from Life Technologies (Grand Island, NY); [14C]chloramphenicol (53 mCi/mmol) and [3H]R5020 (86.7 C/mmol) were purchased from NEN Research Products (Boston, MA), and [3H]E2 (130 Ci/mmol) was purchased from Amersham Life Sciences (Arlington Heights, IL). The human cathepsin D construct (pCD) contained a –365 to –10 insert from the cathepsin D gene promoter ligated into a pBL/TATA/chloramphenicol acetyl transferase (CAT) plasmid derived from pBL/CAT2. The creatine kinase B construct (pCKB) contains a 2.90 kb sequence from the rat CKB gene that was provided by Dr P.Benfield (Dupont, Wilmington, DE) (27). Synthesis of PCB congeners in this laboratory have previously been described (28); 3,3',4,4'-tetraCB was prepared by diazotization of 3,3'-dichlorobenzidine in aqueous hydrochloric acid/sodium nitrite followed by decomposition in cuprous chloride; 3,3',4,4',5-pentaCB and 2,2',5,5'-tetraCB are synthesized by diazo coupling of 3,4-dichloroaniline or 2,5-dichloroaniline with excess 1,2,3-trichlorobenzene or 1,4-dichlorobenzene, respectively (compounds purchased from Aldrich Chemical, Milwaukee, WI). All PCB reaction products were purified by silica gel column or thin-layer chromatography using hexane as a solvent and crystallized from methanol or anisole/methanol to give congeners with purities >98–99% as determined by gas–liquid chromatography. 3,3',4,4',5-PentaCB is formed as a mixture with 2,3,3',4,4'-pentaCB and these isomers are readily separated by column chromatography using silica gel coated with charcoal. All other chemicals and biochemicals were of the highest quality available from commercial sources.

Cell proliferation assay
T47D cells used for proliferation and transient transfection studies were maintained in {alpha} MEM with phenol red and supplemented with 1.2 g/l bicarbonate, 5% FBS, pH 7.4, 10 ml/l antibiotic solution. MCF-7 cells were maintained in MEM with phenol red and supplemented with 10% FBS plus antibiotic/antimycotic solution, 0.035% sodium bicarbonate, 0.011% sodium pyruvate, 0.1% glucose, 0.238% Hepes and 6x10–7% insulin. After treatment with trypsin, cells were seeded in 6 well plates (50 000/well) in DME-F12 supplemented with 5% FBS treated with dextran-coated charcoal (DCC), 1.2 g/l NaHCO3 and 10 ml/l antibiotic solution. After 4 h, medium was changed and cells were treated with the appropriate chemicals dissolved in DMSO. The medium was changed and cells were redosed every 48 h. After 14 days, cells were trypsinized, harvested, centrifuged at 200 g for 5 min at 4°C and resuspended in fresh medium. Cells were counted using a Coulter Z1 cell counter (Coulter Electronics, Hialeah, FL).

CAT assay
Cells were seeded at ~50–60% confluency in 100 mm tissue culture dishes in DME-F12 without phenol red medium supplemented with 5% FBS treated with DCC, 1.2 g/l NaHCO3 and 10 ml/l antibiotic solution. After 24 h, cells were transiently cotransfected with 10 µg pCKB or pCD and 5 µg hER using the calcium phosphate method. After 6 h, cells were shocked with 25% DMSO in PBS. Cells were treated with the appropriate chemicals dissolved in DMSO for 48 h, and equivalent amounts of DMSO were added to the control dishes. After 48 h, cells were washed twice with PBS and scraped from the plates. Cell lysates were prepared in 0.1 ml 0.25 M Tris–HCl, pH 7.8 by three freeze–thaw–sonication cycles. Protein concentrations were determined using bovine serum albumin (BSA) as a standard, and aliquots of cell lysate were incubated with 1 µl [14C]chloramphenicol (52 mCi/mol) and 42 µl of 4 mM acetyl-CoA at 37°C. Ethyl acetate was then added, and the extract was dried, redissolved in 20 µl ethyl acetate and acetylated products resolved by thin-layer chromatography using a 95:5 chloroform:methanol solvent mixture. The percentage of protein conversion into acetylated chloramphenicol was quantitated using the counts per min obtained from the Betagen Betascope 603 Blot Analyzer (Tritech, Annapolis, MD). CAT activity was calculated relative to activity observed in cells treated with DMSO (control), and results are expressed as means ± SE.

Animals
B6C3F1 female mice were purchased from Jackson Laboratories (Bar Harbor, ME) and housed 6–9/cage with ad libitum access to food and water. 3,3',4,4'-TetraCB and 3,3',4,4',5-pentaCB were each dissolved in corn oil with slight warming and the total dose divided into three daily injections. Groups of mice (n = 6–9) received 0.1 ml of 3,3',4,4'-tetraCB (50, 75, 100 or 150 mg/kg) or 3,3',4,4',5-pentaCB (1, 5 or 10 mg/kg) solution or vehicle control i.p. for three days beginning at 21 days of age. Some groups also received 0.02 µg/day of E2 (in corn oil) by i.p. injection on the same three treatment days (days 21–23). The doses of E2 were the minimal effective dose which induced the three uterine responses of interest. Animals were killed by carbon dioxide asphyxiation 20 h after the last treatment and the uteri were quickly removed, cleaned of connective tissue, weighed, nicked and blotted. The uteri were then bisected into right and left halves, each half containing an entire uterine horn.

Progesterone and estrogen receptor binding assays (PR/ER) (27)
The uterine bisections of each treatment group were pooled in an ice cold TESHMo (10 mM Tris–Cl, pH 7.4, 1.5 mM EDTA, 15 mM thioglycerol, 10 mM sodium molybdate) buffer, 1 ml/50 mg tissue. Uteri were homogenized with 3x8 s bursts using a Brinkman/Polytron tissue grinder. Samples were then centrifuged for 45 min at 105 000 g, and the clear supernatant, constituting the cytosol for this experiment, was carefully decanted and immediately used for competitive binding assays. Cytosolic fractions described above were incubated with 20 nM [3H]R5020 in the presence or absence of 2 µM unlabeled R5020 at 4°C. Following an 8 h incubation, samples were placed on ice and treated with 0.1 volume DCC suspension (0.5% dextran:5% charcoal, w/v in TESHMo) for 10 min with gentle shaking. Samples were then centrifuged at 5000 g for 10 min, and the supernatant measured by liquid scintillation counting. PR levels were calculated assuming a 1:1 binding between PR and [3H]R5020. Levels are reported in fmol per uterus. Ten nanomoles of [3H]E2 were used as the radioligand for determining the competitive displacement of [3H]E2 by different concentrations of the test compounds. Uterine cytosol for these studies was obtained from untreated animals.

Human ER{alpha} competitive binding assay
Recombinant human ER{alpha} was obtained from PanVera (Madison, WI) and competitive ER{alpha} binding was determined using the accompanying assay protocol. Briefly, 20 nM [3H]E2 was incubated with varying concentrations of unlabeled E2, 3,3',4,4'-tetraCB, 3,3',4,4',5-pentaCB and 2,2',5,5'-tetraCB at 4°C for 14–16 h. Hydroxylapatite was added to the incubation and vortexed three times over 15 min. A wash buffer (40 mM Tris–base, 100 mM KCl, 1 mM EDTA, 1 mM EGTA) was added to the mixture and centrifuged at 10 000 r.p.m. for 5 min. The supernatant was discarded and the washing repeated twice more. The resulting pellet was resuspended in ethanol, transferred to scintillation vials and counted on a liquid scintillation counter. Activity is reported as % [3H]E2 displaced from the receptor.

Uterine peroxidase assay (UPO)
Uterine bisections from the different treatment groups were pooled and homogenized as described above. Homogenates were centrifuged at 39 000 g at 2°C for 45 min and the resulting pellets were washed and resuspended in 10 mM Tris–Cl buffer containing 0.5 M CaCl2. Extracts were clarified by centrifugation for 45 min at 39 000 g at 2°C. Uterine peroxidase activities of the supernatants were determined as described (29). Each assay mixture (3.0 ml total) contained 1 mM guaiacol and 0.3 mM H2O2 in the extraction buffer. The reaction was started by addition of 1.0 ml of the CaCl2 extract. The initial rate (1 min) of guaiacol oxidation was monitored at {lambda} = 470 nm on a Beckman spectrophotometer. An enzyme unit was defined as the amount of enzyme required to produce an increase of one absorbance unit per min under the assay conditions described. Enzyme activity is expressed per mg extract protein, measured by the method of Bradford (30).

Mammary tumor studies (31)
Forty-nine-day-old female virgin Sprague–Dawley rats were obtained from Harlan (Houston, TX) and allowed to acclimate for 5 days. On day 54, each rat received 20 mg DMBA dissolved in 0.5 ml corn oil (by gavage). Tumors developed 30–75 days after treatment with DMBA and after initial formation of tumors (250–400 mm3), rats were dosed i.p. twice weekly with either vehicle control corn oil (1.6 ml/kg) or 3,3',4,4'-tetraCB in corn oil (1.6 ml/kg) at a dose of 25 mg/kg. Tumor volumes were measured with calipers every other day, and volumes were calculated by the formula (lengthxwidthxdepth)/6{pi} and are expressed as percentage of control. On day 22 (3.5 days after the last injection), rats were killed by asphyxiation, and organ and tumor weights were determined as previously described (31). Livers were weighed and perfused, microsomes were prepared, and ethoxyresorufin O-deethylase (EROD) activity was determined fluorimetrically as previously described (31).

Electrophoretic mobility shift assays
Oligonucleotides were annealed and labeled at the 5' end using T4-polynucleotide kinase and [{gamma}-32P]ATP. Gel electrophoretic mobility shift assays were performed by incubating 50 fmol pure human estrogen receptor (hER) protein (PanVera) in 25 µl of 1x binding buffer (5% glycerol, 1 mM EDTA, 0.5 mM DTT, 100 mM KCl, 20 mM Hepes, pH 8.0), containing 0.2 µM E2 or desired chemicals for 15 min at 4°C, balancing the protein concentration with BSA. After incubation for 15 min at 4°C, 32P-labeled oligonucleotides (50 000 c.p.m.) were added to the reaction mixture in the presence of 500 ng poly[d(I-C)] and incubated for an additional 15 min at 25°C. Excess unlabeled DNA was added before adding 32P-labeled oligonucleotides. Magnesium chloride (5 mM) was added to the mixture and incubated for 15 min at 37°C. The following procedure was used for rat hepatic cytosol. Rat hepatic cytosol was incubated with desired chemicals such that the final concentration of DMSO was 1% (v/v) for 2 h at 25°C. Cytosol (80 µg) was incubated in HEGDK (final concentration of KCl was 100 mM) with 1 µg of poly[d(I-C)] for 15 min. Following the addition of 32P-labeled DRE oligonucleotides (100 000 c.p.m.), the mixture was incubated for additional 15 min at 25°C. Excess unlabeled DRE was added 5 min before adding 32P-labeled DRE to compete for the specific DNA–protein binding. Protein–DNA complexes were resolved on a 5–6% polyacrylamide gel (acrylamide:bisacrylamide ratio, 30:0.8) and run in 1x TBE buffer (0.9 M Tris, 0.09 boric acid, 2 mM EDTA, pH 8.3) at 110 V. Protein–DNA binding was visualized by autoradiography and quantitated by densitometry using the Molecular Dynamics Zero-D software package (Sunnyvale, CA) and a Sharp JX-330 scanner (Sharp, Mahwah, NJ) and subjected to autoradiography using a Kodak X-Omat film (Eastman Kodak, Rochester, NY) for appropriate times at –80°C.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Immature B6C3F1 mouse uterus
The effects of 3,3',4,4'-tetraCB on estrogenic activities in the immature mouse uterus were determined in parallel with 3,3',4,4',5-pentaCB, another coplanar PCB congener that is more potent than 3,3',4,4'-tetraCB in most assays as an AhR agonist (32). The results illustrated in Figure 1Go show that neither 3,3',4,4'-tetraCB nor 3,3',4,4',5-pentaCB competitively displaced [3H]E2 in a competitive binding assay using mouse uterine cytosol; previous studies using the same assay (12) showed that both 2,2',4,6,6'-penta- and 2,2',4,4',6,6'-hexachlorobiphenyl competitively displaced [3H]E2. Similar results were also obtained using recombinant human ER; unlabeled E2 but not 3,3',4,4'-tetraCB, 3,3',4,4',5-pentaCB or 2,2',5,5'-tetraCB competitively displaced [3H]E2 from the human ER (Figure 1Go). The comparative effects of E2 alone (0.02 µg/mouse), different doses of 3,3',4,4'-tetraCB alone or in combination with E2 on uterine wet weight, progesterone receptor (PR) binding and UPO activity were determined in the immature B6C3F1 mouse uterus (Figure 2Go). Hormone/chemicals were administered on three consecutive days (days 21, 22 and 23 of age) and various parameters were determined 20 h after the last treatment. The results show that E2 induced a 3.2-, 4.6- and 8.4-fold increase in uterine wet weight, PR binding and UPO activity, respectively; in contrast, 3,3',4,4'-tetraCB alone did not significantly affect uterine wet weight or PR binding; however, a small but significant dose-independent increase in UPO activity was observed only at doses of 50 and 75 mg/kg, and this type of dose-independent response has been observed for other chemicals (21). In mice cotreated with E2 plus 3,3',4,4'-tetraCB, there was a decrease in E2-induced UPO activity at all four doses and, at the highest dose (150 mg/kg), there was a significant decrease in E2-induced uterine wet weight, PR binding and peroxidase activity. In mice cotreated with E2 and 1, 5 or 10 mg/kg of the more potent AhR agonist 3,3',4,4',5-pentaCB, there was significant inhibition of all three estrogen-induced responses at all doses (Figure 3Go) and this was consistent with the structure-dependent differences in the in vivo potencies of these PCB congeners as AhR agonists (32).



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  		Fig. 1. ER binding assays. (A) Mouse ER. Mouse uterine cytosol was isolated from B6C3F1 mice and competitive ER binding was determined using 10 nM [3H]E2 and different concentrations of unlabeled E2 and PCB congeners as described in the Materials and methods. Unlabeled E2 ({blacksquare}) significantly displaced [3H]E2 in this assay, whereas 3,3',4,4'-tetraCB (•) and 3,3',4,4',5-pentaCB ({square}) were inactive. (B) Human ER. Competitive binding assays were also determined using human ER as described in the Materials and methods. Unlabeled E2 ({blacksquare}) but not 3,3',4,4'-tetraCB (•), 2,2',5,5'-tetraCB ({blacktriangleup}), and 3,3',4,4',5-pentaCB ({square}) competitively displaced [3H]E2 in this assay.

 


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  		Fig. 2. Estrogenic and antiestrogenic activity of 3,3',4,4'-tetraCB in female B6C3F1 mice. (A) Estrogenic activity. Twenty-one-day-old mice were treated with E2 (0.02 µg/day) or different doses of 3,3',4,4'-tetraCB for 3 consecutive days, and 20 h after the last treatment, mice were killed and uterine wet weight, peroxidase activity and PR binding were determined as described in the Materials and methods. E2 significantly induced (*P < 0.05) all three uterine responses and 3,3',4,4'-tetraCB only slightly induced uterine peroxidase activity at the two lower doses (50 and 75 mg/kg). (B) Antiestrogenic activity. The same protocols were used as described in (A) except that mice were cotreated with E2 (0.02 µg/day) plus different doses of 3,3',4,4'-tetraCB. Significant decreases (*P < 0.05) in E2-induced uterine peroxidase activity were observed at all four dose levels and all E2-induced uterine responses were inhibited at the highest dose of 3,3',4,4'-tetraCB (150 mg/kg).

 


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  		Fig. 3. Antiestrogenic activity of 3,3',4,4',5-pentaCB in the mouse uterus. Twenty-one-day-old B6C3F1 mice were cotreated with E2 (0.02 µg/day) plus different doses of 3,3',4,4',5-pentaCB as described in Figure 2Go. Significant (*P < 0.05) inhibition of uterine wet weight, peroxidase activity and PR binding were observed at all three doses.

 
Antitumorigenic activity of 3,3',4,4'-tetraCB in DMBA-induced rat mammary tumors
The antiestrogenic activity of 3,3',4,4'-tetraCB was also investigated in the DMBA-induced mammary tumor model in female Sprague–Dawley rats. Fifty-four day-old rats were initiated with DMBA (20 mg/rat) and after 30–60 days, 3,3',4,4'-tetraCB (25 mg/kg, 2x weekly) was administered to animals after initial observation of mammary tumors (31). The time-dependent effects of 3,3',4,4'-tetraCB on tumor volume over the treatment period is summarized in Figure 4Go and shows that 3,3',4,4'-tetraCB inhibited mammary tumor growth (volume and weight), decreased spleen weight, increased liver/body weight ratios and induced CYP1A1-dependent hepatic microsomal EROD activity (Table IGo). The latter response is induced by coplanar PCBs and fully chlorinated AhR agonists in rodent liver (32).



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  		Fig. 4. Antitumorigenic activity of 3,3',4,4'-tetraCB. Female Sprague–Dawley rats were initiated with DMBA and after initial tumors were observed, animals were treated with corn oil ({circ}) or 3,3',4,4'-tetraCB (•) in corn oil (25 mg/kg; 2x weekly) for 3 weeks and killed as described in the Materials and methods. 3,3',4,4'-TetraCB significantly inhibited tumor growth as summarized in Table IGo.

 

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  		Table I. Effect of 3,3',4,4'-tetraCB on tumor volume and weight, organ weights and hepatic microsomal EROD activity
 
Activities of 3,3',4,4'-tetraCB and 3,3',4,4',5-pentaCB in human breast cancer cells
The results in Figure 5Go compare the concentration-dependent effects of 3,3',4,4'-tetraCB and 3,3',4,4',5-pentaCB alone and in combination with E2 on proliferation of MCF-7 and T47D human breast cancer cells. No significant induction of cell proliferation was observed for either compound and this was in direct contrast to results of previous studies which showed that 3,3',4,4'-tetraCB induced MCF-7 and ZR-75–1 breast cancer cell proliferation with a maximal response observed at a concentration of 1 nM (14). Cotreatment of T47D and MCF-7 cells with 1 nM E2 and 3,3',4,4'-tetraCB or 3,3',4,4'5-pentaCB resulted in significant inhibition of E2-induced proliferation by both compounds. The effects of 3,3',4,4'-tetraCB and 3,3',4,4',5-pentaCB alone and in combination with E2 were also determined in MCF-7 and T47D cells transiently transfected with pCKB and pCD constructs containing promoter inserts from the creatine kinase B and cathepsin D genes, respectively (Figures 6 and 7GoGo). The PCB congeners alone (10 µM) did not induce (or decrease) CAT reporter gene activity and induction was not observed at lower concentrations of these compounds; in contrast, 1 nM E2 induced a 3.0- and 2.7-fold increase in CAT activity in T47D and MCF-7 cells transfected with pCD and a 3.5- and 7.0-fold increase in CAT activity in T47D and MCF-7 cells, respectively, transfected with pCKB. In cells transfected with pCKB and cotreated with 10 µM 3,3',4,4'-tetraCB or 3,3,4,4',5-pentaCB plus E2, there was a significant inhibition of the E2 induced response in MCF-7 cells but not in T47D cells (Figure 6Go). In contrast, both PCB congeners inhibited E2-induced CAT activity in both cell lines transfected with pCD (Figure 7Go). As a positive control for these experiments, it was shown that 1 µM ICI 182,780 significantly inhibited reporter gene activity induced by E2 in MCF-7 and T47D cells transfected with pCKB (Figure 6Go) or pCD (Figure 7Go).



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  		Fig. 5. Effects of 3,3',4,4'-tetraCB and 3,3',4,4',5-pentaCB on proliferation of T47D (a) and MCF-7 (b). T47D and MCF-7 cells were treated with 1 nM E2 alone, DMSO (vehicle control), different concentrations of 3,3',4,4'-tetraCB or 3,3',4,4',5-pentaCB alone or in combination with E2 as described in the Materials and methods. In T47D cells (a), both PCB congeners alone significantly inhibited cell growth (aP < 0.05) and in combination with E2 significantly inhibited hormone-induced cell proliferation (bP < 0.05). In MCF-7 cells, 3,3',4,4'-tetraCB and 3,3',4,4',5-pentaCB also inhibited E2-induced cell proliferation (bP < 0.05) but these compounds alone did not affect cell growth. Higher concentrations of both PCB congeners (>10 µM) were cytotoxic and markedly affected cell attachment.

 


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  		Fig. 6. Estrogenic and antiestrogenic activities of PCB congeners in breast cancer cells transfected with pCKB. T47D (a) or MCF-7 (b) cells were transiently transfected with pCKB and treated with DMSO, 1 nM E2, 1 µM ICI 182,780, 10 µM 3,3',4,4'-tetraCB or 3,3',4,4',5-pentaCB alone and E2 plus PCB congeners or ICI 182,780. CAT activity was then determined as described in the Materials and methods. Results are expressed as means ± SE for three separate determinations for each treatment group. E2 alone significantly (aP < 0.05) induced CAT activity and ICI 182,780 significantly inhibited (bP < 0.05) E2-induced CAT activity in both cell lines. Ten micromoles of 3,3',4,4'-tetraCB and 3,3',4,4',5-pentaCB alone were not estrogenic in both cell lines and both congeners significantly (bP < 0.05) inhibited E2-induced activity in MCF-7 but not T47D cells.

 


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  		Fig. 7. Estrogenic and antiestrogenic activity of PCB congener in breast cancer cells transfected with pCD. The assay procedures and treatment groups were identical to those described in Figure 6Go except that cells were transfected with pCD. E2 alone significantly (aP < 0.05) induced CAT activity, whereas the PCB congeners were not estrogenic in T47D (a) and MCF-7 (b) cells. In the combined treatment groups 3,3',4,4'-tetraCB, 3,3',4,4',5-pentaCB and ICI 182,780 significantly (bP < 0.05) inhibited E2-induced activity in both cell lines. Results are presented as means ± SE for three separate determinations for each treatment group.

 
Gel mobility shift assays
The results in Figure 8AGo compare the ligand-dependent transformation of rat hepatic cytosol and binding to [32P]DRE in a gel mobility shift assay. Treatment of cytosol with 1 nM TCDD induced formation of a complex retarded band (lane 3) compared to DMSO (lane 2) and intensity of this band was decreased after competition with excess unlabeled wild-type DRE (lane 4) but not mutant DRE (lane 5). The potent AhR agonist 3,3',4,4',5-pentaCB induced retarded band formation at all concentrations (200–0.2 µM, lanes 12–15), whereas 3,3',4,4'-tetraCB was active only at the highest concentration (200 µM, lane 6). In contrast, 2,2',5,5'-tetraCB did not induce transformation of rat hepatic cytosol (lanes 9–11) and this was consistent with the negligible AhR agonist activity of this compound (32). Nesaretnam et al. (14) previously reported that 3,3',4,4'-tetraCB induced in vitro translated human ER binding to [32P]ERE in a gel mobility shift assay; however, intensity of the retarded band was inversely related to ligand concentration and the estrogenic hydroxy-PCB metabolite of 3,3',4,4'-tetraCB was less active than the parent hydrocarbon in this assay. The results in Figure 8BGo also show that E2 induces transformation of human ER to form a specifically bound retarded band in a gel mobility shift assay (lanes 2 and 3). However, results obtained with 3,3',4,4'-tetraCB (lanes 6–10) and 2,2',5,5'-tetraCB (lanes 11–15) indicate that transformation by these compounds was both structure- and concentration-independent indicating that this response was non-specific. Similar results were also obtained for 3,3',4,4',5-pentaCB (data not shown).




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  		Fig. 8. Gel mobility shift assays. (A) Ligand-induced transformation of rat hepatic cytosol and binding to [32P]DRE. Rat liver cytosol was incubated with 10 nM TCDD and different concentrations of 3,3',4,4'-tetraCB, 2,2',5,5'-tetraCB and 3,3',4,4',5-pentaCB and analyzed by gel mobility shift assays as described in the Materials and methods. Ten nM TCDD induced formation of a retarded band (bound DNA R) (lane 3) which was decreased by competition with unlabeled wild-type DRE (lane 4) but not mutant DRE. Transformation of cytosol with 3,3',4,4'-tetraCB (200 to 2 µM; lanes 6–8) was observed only at the highest concentration, whereas 3,3',4,4',5-pentaCB (200 to 0.2 µM; lanes 12–15) was active at all concentrations. In contrast, 2,2',5,5'-tetraCB (200 to 2 µM; lanes 9–11) was inactive at all concentrations. (B) Ligand-induced binding of ER to [32P]ERE. Recombinant ER (50 fmol) was incubated with [32P]ERE, E2, 3,3',4,4'-tetraCB or 2,2',5,5'-tetraCB and formation of an ER-ERE retarded band (bound DNA R) was determined by gel mobility shift assay as described in the Materials and methods. Although a specifically bound retarded band was observed in the absence of ligand (lane 2), 200 nM E2 caused a 3- to 4-fold increase in retarded band intensity (lane 3) that was decreased after competition with excess unlabeled wild-type ERE (lane 4) but not mutant ERE (lane 5) oligonucleotides. Both 3,3',4,4'-tetraCB (200–0.02 µM; lanes 6–10) and non-estrogenic 2,2',5,5'-tetraCB (200–0.02 µM; lanes 11–15) induced a concentration-independent formation of the retarded band. This lack of specificity was observed in at least three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
3,3',4,4'-TetraCB binds the AhR with moderate to low affinity and has been extensively characterized as one of a series of coplanar PCB congeners that exhibit AhR agonist activities in multiple in vivo and in vitro assay systems (32). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent AhR agonist and studies in several laboratories have demonstrated that TCDD and related compounds inhibit E2-induced responses in the rodent uterus and mammary tissue and in human breast cancer cells in culture (reviewed in refs 25,26). For example, TCDD inhibits E2-induced uterine wet weight increase, peroxidase activity and PR binding in the rodent uterus (33–39) and inhibits (i) spontaneous age-dependent mammary and uterine tumor formation in female Sprague–Dawley rats; (ii) carcinogen-induced mammary tumor formation and growth in the same rat strain; and (iii) mammary tumor growth in B6C3F1 mice bearing MCF-7 cell xenografts (40–43). The indirect antiestrogenic activity of TCDD has been extensively characterized in MCF-7 cells using cathepsin D and pS2 genes as models, one of the mechanisms of AhR-ER crosstalk involved interaction of the AhR complex with functional pentanucleotide sequences (GCGTG) in the pS2 and cathepsin D gene promoters that corresponded to the core sequence of the dioxin responsive element (44,45).

Nesaretnam et al. recently reported that 3,3',4,4'-tetraCB bound to the ER and induced estrogenic responses in MCF-7 cells and the immature female mouse uterus and enhanced mammary tumor formation in the DMBA-induced rat mammary tumor model (14,46). These results were in conflict with studies from several laboratories demonstrating that coplanar PCBs, TCDD and other AhR agonists exhibited antiestrogenic activities (24,25,32). Therefore, we have reinvestigated the estrogenic and antiestrogenic responses of both 3,3',4,4'-tetraCB and 3,3',4,4',5-pentaCB, a structurally related congener that has been characterized as a more potent AhR agonist than 3,3',4,4'-tetraCB (32).

Our results show that neither PCB congener competitively bound the mouse uterine nor human ER (Figure 1Go), and the mouse ER binding assay was sufficiently sensitive to detect competitive binding of other weakly estrogenic PCBs (12). Moreover, in the immature mouse uterine assay system, both 3,3',4,4'-tetraCB and 3,3',4,4',5-pentaCB inhibited E2-induced uterine wet weight increase, peroxidase activity and PR binding; 3,3',4,4',5-pentaCB was a more potent antiestrogen than 3,3',4,4'-tetraCB in these in vivo studies and this corresponded to their order of potency for other AhR-mediated responses (12,32). Results of in vitro ER (mouse and human) binding and in vivo studies in the immature mouse uterus contrasted to the previous report showing that 3,3',4,4'-tetraCB bound human ER and induced uterine wet weight increase in 25–28-day-old CD-1 mice (14). However, it was previously reported that high doses of 3,3',4,4'-tetraCB inhibited E2-induced uterine wet weight increase in 25–28-day-old CD-1 mice and in younger animals (21–25 days old) estrogenic activity was not observed for 3,3',4,4'-tetraCB (14). These results might explain, in part, differences observed with CD-1 mice (14) compared with results of this study where both coplanar PCBs were not ER agonists but exhibited antiestrogenic activities in the 21–25-day-old B6C3F1 mouse uterus (Figures 2 and 3GoGo).

Nesaretnam et al. also reported an unusual concentration-independent effect of 3,3',4,4'-tetraCB on growth of E2-responsive MCF-7 and ZR-75 breast cancer cells (14); 1 nM 3,3',4,4'-tetraCB induced proliferation of both cell lines whereas both higher (0.1 and 0.01 nM) and lower (10–1000 nM) concentrations exhibited minimal estrogenic activities. This inverted U-shaped dose–response curve in breast cancer cells has also been reported for increased prostate weight of adult mice after in utero exposure to the weakly estrogenic industrial compound bisphenol A (47). However, it was surprising that despite the low reported binding affinity of 3,3',4,4'-tetraCB for the ER, the mitogenic potency was similar to that of E2 and this was also reported for some gene expression assays (14). In contrast, our results showed that 1–1000 nM concentrations of 3,3',4,4'-tetraCB and 3,3',4,4',5-pentaCB did not induce proliferation of MCF-7 or T47D cells (Figure 5Go). Moreover, in cells cotreated with E2 plus PCB congeners there was a significant decrease in hormone-induced proliferation and this complemented previous data showing that other structural classes of AhR agonists including polycyclic aromatic hydrocarbons (PAHs) and hetero-PAHs also exhibited antiestrogenic activity in this assay (48–50). 3,3',4,4'-TetraCB and 3,3',4,4',5-pentaCB exhibit similar IC50 values for binding to the AhR; however, for most studies the penta congener is more potent due to more rapid metabolism of 3,3',4,4'-tetraCB (32). In contrast, results of the in vitro studies (Figures 5–7GoGoGo) show that both compounds were approximately equipotent and this may be related to the relatively low rate of PCB metabolism in MCF-7 cells.

With one exception, both coplanar PCB congeners inhibited E2-induced reporter gene activity in MCF-7 and T47D cells transfected with pCD or pCKB (Figures 6 and 7GoGo) and in these same transactivation assays no ER agonist activity was observed for 10 µM (and lower doses; data not shown) 3,3',4,4'-tetraCB or 3,34,4',5-pentaCB alone. The failure to observe AhR-mediated inhibition of CAT activity in T47D cells transfected with pCKB (Figure 6Go) has also been observed for other AhR agonists including TCDD (data not shown) and deletion constructs derived from the CKB gene promoter are being utilized to further investigate cell-specific differences (MCF-7 versus T47D) in ER–AhR crosstalk.

Nesaretnam et al. (14) previously showed that 3,3',4,4'-tetraCB induced ER transformation as detected by gel mobility shift assays. However, their results showed that intensity of the retarded band was inversely related to ligand concentration (10–6 M > 10–5 M) and a 10–5 M concentration of an estrogenic hydroxy-PCB metabolite gave a weaker band than the parent hydrocarbon. Our results showed concentration-independent transformation of ER and ERE binding for several concentrations of 3,3',4,4'-tetraCB (Figure 8BGo) and these data were similar to results reported for 3,3',4,4'-tetraCB in the same assay (14). Moreover, similar responses (Figure 8BGo) were also observed for 3,3',4,4',5-pentaCB (an AhR agonist) and a non-estrogenic PCB congener, 2,2',5,5'-tetraCB. This lack of compound specificity for the gel mobility shift assay suggests that this is not a diagnostic response for determining estrogenic activity of PCB congeners.

A recent study also reported that 3,3',4,4'-tetraCB enhanced the rate of mammary tumor formation in DMBA-induced female Sprague–Dawley rats maintained on a high or low-fat diet (46). However, the PCB was co-administered with DMBA and, therefore, increased mammary tumor formation was probably related to effects of 3,3',4,4'-tetraCB on metabolic activation of DMBA since 3,3',4,4'-tetraCB induces both phase I and phase II drug-metabolizing enzymes (32). In contrast, our results (Figure 4Go) showed that 3,3',4,4'-tetraCB inhibited mammary tumor growth when administered after initial tumor formation and this was consistent with the antitumorigenic activity of other AhR agonists in rodent assays for mammary cancer (40–43). Our results do not conflict with the previous report (46) due to differences in the timing of compound administration; however, it is also clear that the overall evaluation of the tumorigenic/antitumorigenic activities of 3,3',4,4'-tetraCB should consider both the enhancing and protective effects of this compound in mammary carcinogenesis.

Results of this study demonstrate that two structurally related coplanar PCB congeners do not exhibit ER agonist activity in a series of in vitro and in vivo bioassays. Moreover, both 3,3',4,4'-tetraCB and 3,3',4,4',5-pentaCB inhibited a diverse spectrum of E2-induced responses that are consistent with previous reports showing crosstalk between the AhR and ER signaling pathways (25,26,44,45). It is difficult to rationalize differences between results of this study and those reported by Nesaretnam et al. (14,46); however, their observation of high potency for 3,3',4,4'-tetraCB and an inverted U-shaped dose–response curve for cell proliferation suggests that unknown impurities or metabolites may play a role in this response.


   Notes
 
1 To whom correspondence should be addressed Email: ssafe{at}cvm.tamu.edu Back


   Acknowledgments
 
The financial assistance of the National Institutes of Health (ES04917, ES04176 and ES09106) and the Texas Agricultural Experiment Station is gratefully acknowledged. S.S. is a Sid Kyle Professor of Toxicology.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 28, 1998; revised September 10, 1998; accepted September 11, 1998.


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D. Bagga, K. H. Anders, H.-J. Wang, E. Roberts, and J. A. Glaspy
Organochlorine Pesticide Content of Breast Adipose Tissue From Women With Breast Cancer and Control Subjects
J Natl Cancer Inst, May 3, 2000; 92(9): 750 - 753.
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