Fig. 1. ER binding assays. (A) Mouse ER 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 (▪) significantly displaced [³H]E2 in this assay, whereas 3,3′,4,4′-tetraCB (•) and 3,3′,4,4′,5-pentaCB (□) were inactive. (B) Human ER. Competitive binding assays were also determined using human ER as described in the Materials and methods. Unlabeled E2 (▪) but not 3,3′,4,4′-tetraCB (•), 2,2′,5,5′-tetraCB (▴), and 3,3′,4,4′,5-pentaCB (□) competitively displaced [³H]E2 in this assay. From: 3,3′,4,4′-Tetrachlorobiphenyl exhibits antiestrogenic and antitumorigenic activity in the rodent uterus and mammary cells and in human breast cancer cells Carcinogenesis. 1999;20(1):115-123. doi:10.1093/carcin/20.1.115 Carcinogenesis | © Oxford University Press

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). From: 3,3′,4,4′-Tetrachlorobiphenyl exhibits antiestrogenic and antitumorigenic activity in the rodent uterus and mammary cells and in human breast cancer cells Carcinogenesis. 1999;20(1):115-123. doi:10.1093/carcin/20.1.115 Carcinogenesis | © Oxford University Press

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 2. Significant (*P < 0.05) inhibition of uterine wet weight, peroxidase activity and PR binding were observed at all three doses. From: 3,3′,4,4′-Tetrachlorobiphenyl exhibits antiestrogenic and antitumorigenic activity in the rodent uterus and mammary cells and in human breast cancer cells Carcinogenesis. 1999;20(1):115-123. doi:10.1093/carcin/20.1.115 Carcinogenesis | © Oxford University Press

Fig. 4. Antitumorigenic activity of 3,3′,4,4′-tetraCB 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 (○) or 3,3′,4,4′-tetraCB (•) in corn oil (25 mg/kg; 2× 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 I. From: 3,3′,4,4′-Tetrachlorobiphenyl exhibits antiestrogenic and antitumorigenic activity in the rodent uterus and mammary cells and in human breast cancer cells Carcinogenesis. 1999;20(1):115-123. doi:10.1093/carcin/20.1.115 Carcinogenesis | © Oxford University Press

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. From: 3,3′,4,4′-Tetrachlorobiphenyl exhibits antiestrogenic and antitumorigenic activity in the rodent uterus and mammary cells and in human breast cancer cells Carcinogenesis. 1999;20(1):115-123. doi:10.1093/carcin/20.1.115 Carcinogenesis | © Oxford University Press

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. From: 3,3′,4,4′-Tetrachlorobiphenyl exhibits antiestrogenic and antitumorigenic activity in the rodent uterus and mammary cells and in human breast cancer cells Carcinogenesis. 1999;20(1):115-123. doi:10.1093/carcin/20.1.115 Carcinogenesis | © Oxford University Press

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 6 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. From: 3,3′,4,4′-Tetrachlorobiphenyl exhibits antiestrogenic and antitumorigenic activity in the rodent uterus and mammary cells and in human breast cancer cells Carcinogenesis. 1999;20(1):115-123. doi:10.1093/carcin/20.1.115 Carcinogenesis | © Oxford University Press

Fig. 8. Gel mobility shift assays 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. From: 3,3′,4,4′-Tetrachlorobiphenyl exhibits antiestrogenic and antitumorigenic activity in the rodent uterus and mammary cells and in human breast cancer cells Carcinogenesis. 1999;20(1):115-123. doi:10.1093/carcin/20.1.115 Carcinogenesis | © Oxford University Press

Fig. 8. Gel mobility shift assays 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. From: 3,3′,4,4′-Tetrachlorobiphenyl exhibits antiestrogenic and antitumorigenic activity in the rodent uterus and mammary cells and in human breast cancer cells Carcinogenesis. 1999;20(1):115-123. doi:10.1093/carcin/20.1.115 Carcinogenesis | © Oxford University Press