Toxaphene Mixture in the ... - CiteSeerX

0013-7227/97/$03.00/0 Endocrinology Copyright © 1997 by The Endocrine Society

Vol. 138, No. 4 Printed in U.S.A.

Estrogenic Activity of a Dieldrin/Toxaphene Mixture in the Mouse Uterus, MCF-7 Human Breast Cancer Cells, and Yeast-Based Estrogen Receptor Assays: No Apparent Synergism* KAVITA RAMAMOORTHY, FAN WANG, I-CHEN CHEN, JOHN D. NORRIS, DONALD P. MCDONNELL, LINDA S. LEONARD, KEVIN W. GAIDO, WAYNE P. BOCCHINFUSO, KENNETH S. KORACH, AND STEPHEN SAFE† Veterinary Physiology and Pharmacology (K.R., F.W., I-C.C, S.S), Texas A&M University, College Station, Texas 77843-4466; Department of Pharmacology (J.D.N., D.P.M.), Duke University Medical School, Durham, North Carolina 27709; Chemical Industry Institute of Toxicology (L.S.L., K.W.G.), Research Triangle Park, North Carolina 27709; and Laboratory of Reproductive and Developmental Toxicology (W.P.B., K.S.K.), National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 ABSTRACT The estrogenic activity of dieldrin, toxaphene, and an equimolar mixture of both compounds (dieldrin/toxaphene) was investigated in the 21-day-old B6C3F1 mouse uterus, MCF-7 human breast cancer cells, and in yeast-based reporter gene assays. Treatment of the animals with 17b-estradiol (E2) (0.0053 kg/day 33) resulted in a 3.1-, 4.8-, and 7.8-fold increase in uterine wet weight, peroxidase activity, and progesterone receptor binding, respectively. In contrast, treatment with 2.5, 15 and 60 mmol/kg (33) doses of toxaphene, dieldrin, or dieldrin/toxaphene (equimolar) did not significantly induce a dosedependent increase in any of the E2-induced responses. The organochlorine pesticides alone and the binary mixture did not bind to the mouse uterine estrogen receptor (ER) in a competitive binding assay using [3H]E2 as the radioligand. In parallel studies, estrogenic activities were determined in MCF-7 cells by using a cell proliferation assay and by determining induction of chloramphenicol acetyl transferase (CAT) activity in MCF-7 cells transiently transfected with plasmids containing estrogen-responsive 59-promoter regions from the rat creatine kinase B and human cathepsin D genes. E2 caused a 24-fold increase in CAT activity in MCF-7 cells transiently transfected with creatine kinase B and a 3.8-fold increase in cells transiently transfected with the human cathepsin D construct. Treatment of MCF-7 cells with dieldrin, toxaphene, or an equimolar mixture of dieldrin plus toxaphene (1028–1025 M) did not significantly induce cell proliferation or CAT activity in the transient transfection experiment with both plasmids. The relative competitive binding of the organochlorine pesticides was determined by incubating MCF-7 cells with

1029 M [3H]E2 in the presence or absence of 2 3 1027 M unlabeled E2 (to determine nonspecific binding), toxaphene (1025 M), dieldrin (1025 M), and equimolar concentrations of the dieldrin plus toxaphene mixture (1025 M). The binding observed for [3H]E2 in the whole cell extracts was displaced by unlabeled E2, whereas the organochlorine pesticides and binary mixture exhibited minimal to nondetectable competitive binding activity. E2 caused a 5000-fold induction of b-galactosidase (b-gal) activity in yeast transformed with the human ER and a double estrogen responsive element upstream of the b-gal reporter gene. Treatment with 1026–1024 M chlordane, dieldrin, toxaphene, or an equimolar mixture of dieldrin/toxaphene did not induce activity, whereas 1024 M endosulfan caused a 2000-fold increase in b-gal activity. Diethylstilbestrol caused a 20-fold increase in activity in yeast transformed with the mouse ER and a single estrogen responsive element upstream of the b-gal reporter gene. Dieldrin, chlordane, toxaphene, and endosulfan induced a 1.5- to 4-fold increase in activity at a concentration of 2.5 3 1025 M. Synergistic transactivation was not observed for any equimolar binary mixture of the pesticides at concentrations of either 2.5 3 1025 M or 2.5 3 1024 M. The results of this study demonstrate that for several estrogen-responsive assays in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based reporter gene assays, the activities of both dieldrin and toxaphene were minimal, and no synergistic interactions were observed with a binary mixture of the two compounds. (Endocrinology 138: 1520 –1527, 1997)

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compounds such as 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene, other isomeric DDT/1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene analogs, polychlorinated biphenyls (PCBs), hydroxy-PCBs, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and related halogenated aromatic hydrocarbons (1, 2). Many of the organochlorine compounds, some phthalates, and industrial phenolics (bisphenol-A and nonylphenol) exhibit estrogenic activity (3–10), and it has been hypothesized that xenoestrogens may play a role in wildlife reproduction problems, decreased male reproductive capacity, and the increased incidence of breast cancer in women (1, 11–13). The hypothesized role of xenoestrogens in human disease has been chal-

OLBORN and co-workers (1) have summarized results from several studies reporting developmental effects of endocrine-disrupting chemicals on some wildlife populations. Environmental contaminants that modulate endocrine response pathways include persistent organochlorine Received October 16, 1996. Address all correspondence and requests for reprints to: Stephen Safe, Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466. * This work was supported by NIH Grants ES-04917 and DK-48807, the Chemical Manufacturers Association, and the Texas Agricultural Experiment Station, all of which are gratefully acknowledged. † A Sid Kyle Professor of Toxicology.

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lenged (14 –17), and further research is required to substantiate or disprove the hypothesis. A recent study by Arnold and co-workers (18) reported synergistic interactions of binary mixtures of endosulfan, dieldrin, toxaphene, and chlordane in competitive estrogen receptor (ER)-binding assays and in an estrogen-responsive assay in yeast. Less dramatic synergistic interactions between two weakly estrogenic hydroxy PCB congeners were observed also in the yeast assay and in human endometrial cancer cells. Synergistic interactions between estrogenic chemicals have been reported also in other studies using a single concentration of several compounds and their reconstituted mixtures (6, 10, 19); however, the study by Arnold and coworkers (18) reported dramatic synergistic interactions (up to 1600-fold) by comparing the concentration-dependent effects of individual compounds and their binary mixtures. Based on these data, it was suggested that the estrogenic potency of some environmental chemicals, when tested singly, may be underestimated (18). This study has used two compounds, dieldrin and toxaphene, which gave synergistic responses as a binary mixture in the yeast and ER-binding assays in the study reported by Arnold and coworkers (18). The results of this study show that in the mouse uterus, interactions of dieldrin and toxaphene in ER-binding and functional assays were not synergistic; moreover, similar results were observed in MCF-7 human breast cancer cells and yeast-based reporter gene assays using both the mouse and human ER. Thus, the synergistic interactions of weakly estrogenic environmental chemicals are not universally observed in estrogen-responsive assays, and this should be considered in hazard assessment of these environmental contaminants. Materials and Methods Chemicals and biochemicals Dieldrin, endosulfan, chlordane, and toxaphene (technical) were purchased from Chem-Service (West Chester, PA). MCF-7 human breast cancer cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD). DMEM nutrient mixture F-12 Ham (DME/F-12) without phenol red, PBS, acetyl CoA, 17b-estradiol (E2), and 100X antibiotic/antimycotic solution were purchased from Sigma Chemical Company (St. Louis, MO). FBS was obtained from Intergen (Purchase, NY). MEM was purchased from Life Technologies (Grand Island, NY); [14C]chloramphenicol (53 mCi/mmol) and [3H]R5020 (86.7 Ci/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 (CATH-CAT) construct contains a cathepsin D promoter insert (2365 to 210) (20) ligated into a pBL/ TATA/chloramphenicol acetyl transferase (CAT) plasmid derived from pBL/CAT2. The creatine kinase B (CKB-CAT) construct contains a 2.9kilobase rat CKB-CAT construct provided by Dr. P. Benfield (Dupont Corp.) (21). All other chemicals and biochemicals were of the highest quality available from commercial sources.

Animals B6C3F1 female mice were bred in an onsite animal facility and housed 6 to 9 per cage with ad libitum access to food and water. Test compounds were dissolved in corn oil with slight warming, and mice (n 5 6 –9 per group) received 0.1 ml corn oil (control), E2 at a dose of 0.0053 mmol/ kgzday, toxaphene (2.5, 15, 60, or 275 mmol/kg), dieldrin, or an equimolar toxaphene-dieldrin mixture. The mixture was derived by combining equal molar combinations of toxaphene and dieldrin. The solutions (0.1 ml per animal) were administered ip for 3 consecutive days starting at

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21 days of age. Animals were killed by CO2 asphyxiation 20 h after the last treatment, and uteri were excised, blotted, and weighed. The uteri were then placed in ice-cold buffer and bisected, such that each half contained an entire uterine horn.

Cell proliferation assay MCF-7 cells (50,000) were seeded in six-well multiwell plates in media containing 2 ml DME/F12 without phenol red, supplemented with 5% FBS treated with dextran-coated charcoal (FBS-DCC), 2.2 g/liter sodium bicarbonate, 10 mg/liter apotransferrin, and 20 mg/ml BSA. After 24 h, the media was changed, and the cells were treated with 1029 m E2, 1027–1025 m concentrations of toxaphene, dieldrin, or dieldrin plus toxaphene (equimolar concentrations). The media and chemical treatment were changed every 2 days, and the total duration of the proliferation experiments was 11 days. Cells were then trypsinized, washed with media, resuspended in 1 ml of media, and counted using a Coulter counter, as previously described (22). All determinations were carried out in triplicate, and results are expressed as means 6 sd.

Transient transfection assays MCF-7 cells for proliferation and transient transfection studies were maintained in MEM with phenol red and supplemented with 10% FCS plus antibiotic/antimycotic solution, 0.035% sodium bicarbonate, 0.011% sodium pyruvate, 0.1% glucose, 0.238% HEPES, and 6 3 1027% insulin. MCF-7 cells were then trypsinized, reseeded in 100-mm petri dishes with 10 ml of phenol red-free DME/F-12 medium plus 5% charcoal-stripped FBS, and grown until 50 – 60% confluent. About 3 h before transfection, medium was replaced with 5 ml charcoal-stripped DME/ F-12 medium. Cells in each petri dish were transfected with 1 ml transfection cocktail containing either 10 mg CKB-CAT plus 5 mg hER or 5 mg CATH-CAT plus 4 mg hER, 50 ml of 2.5 m CaCl2, and 500 ml HBS (pH 7.05). After incubation for 14 to 16 h at 37 EC, cells were washed once with 5 ml PBS and treated with test chemicals in 10 ml DCC-stripped DME/F-12 medium. After 48 h, cells were washed once with 5 ml PBS and harvested by scrapping. Cells were lysed in 200 ml of 0.25 m Tris-Cl (pH 7.6) by three cycles of freezing in liquid nitrogen for 2 min, thawing at 37 EC for 2 min, sonication for 3 min, and vortexing for 1 min. Cell debris was pelleted, and the protein concentration in the supernatant was determined (23) using BSA as standard. An aliquot of cell lysate equal to 80 mg protein was diluted to 120 ml with 0.25 m Tris-Cl (pH 7.6) and incubated with 1 ml [14C]chloramphenicol (53 mCi/mmol) and 42 ml of 4 mm acetyl CoA for 6 h at 37 EC. The reaction was stopped by vortexing with 700 ml ethyl acetate. The extract (600 ml) was dried, redissolved in 20 ml ethyl acetate, and the acetylated products resolved by TLC (Whatman Lab Sales, Hillsboro, OR) using a 95:5 chloroform: methanol solvent mixture. The percent protein conversion into acetylated chloramphenicol was quantitated using the counts/minute obtained from the Betagen Betascope 603 blot analyzer (Intelligenetics, Inc., Mountain View, CA). The TLC plates were subjected to autoradiography using a Kodak X-Omat film (Eastman Kodak Co., Rochester, NY) for about 20 h.

Progesterone (PR) and ER-binding assays 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 3 3 8-sec bursts using a Brinkman/Polytron tissue grinder (Brinkman Instruments Co., Westbury, NY). Samples were then centrifuged for 45 min at 105,000 3 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 mM unlabeled R5020 at 4 EC. After an 8-h incubation, samples were placed on ice and treated with 0.1 vol DCC suspension (0.5% dextran:5% charcoal, wt/vol in TESHMo) for 10 min. Samples were then centrifuged at 5,000 3 g for 10 min, and the supernatant was measured by liquid scintillation counting. PR levels were calculated assuming a 1:1 binding between PR and [3H]R5020. Levels are reported in femtomoles per uterus. [3H]E2 (1028 m) was used as a radioligand for determining the competitive displacement of [3H]E2 by

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different concentrations of the test compounds. Uterine cytosol for ER binding was obtained from untreated animals.

Uterine peroxidase (UPO) assay Uterine bisections were pooled into treatment groups and homogenized as described above. Homogenates were centrifuged at 39,000 3 g at 2 EC for 45 min, and the resulting pellet was washed and resuspended in 10 mm Tris-Cl buffer containing 0.5 m CaCl2. The extract was clarified by centrifugation for 45 min at 39,000 3 g at 2 EC. UPO activity of the supernatant was determined as described (24). The assay mixture (3.0 ml total) contained 13 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 l 5 470 nm on a Beckman spectrophotometer (Beckman Instruments Inc., Fullerton, CA). An enzyme unit was defined as the amount of enzyme required to produce an increase of 1 absorbance U per min under the assay conditions described. Enzyme activity is expressed per milligram of extract protein, measured by the method of Bradford (23).

ER binding in MCF-7 cells A suspension of MCF-7 cells in 10 ml serum-free media was incubated with 1029 m [3H]E2 alone or in the presence of 2 3 1027 m unlabeled E2, 25 10 m toxaphene, 1025 m dieldrin, or 1025 m dieldrin/toxaphene mixture for 1 h at 37 EC. Cells were harvested by centrifugation and washed with PBS (20 ml 32). Cells were pelleted by centrifugation, resuspended in 2 ml HED buffer (25 mm HEPES, 1.5 mm EDTA, 1 mm dithiothreitol, pH 7.6), incubated on ice for 10 min, and vigorously homogenized using a teflon pestle/drill apparatus. The homogenate was transferred to a centrifuge tube, rinsed with 2 ml HED buffer, and centrifuged at 1500 3 g at 4 EC for 15 min, and the supernatant was collected and saved. The pellet was resuspended in 1 ml HEGDK (25 mm HEPES, 1.5 mm EDTA, 10% glycerol, 1 mm dithiothreitol, 0.5 m KCl; pH 7.6) and incubated on ice for 1 h with gentle shaking. After centrifugation at 105,000 3 g for 1 h at 4 EC, the supernatant was collected and combined with the supernatant noted above. Samples were then placed on ice and treated with an 0.1 vol DCC suspension (0.5% dextran, 5% charcoal, wt/vol in TESHMo) for 10 min. Samples were then centrifuged at 5,000 3 g for 10 min, and the entire supernatant was measured for radioactivity by liquid scintillation counting. The results are expressed as means 6 se for three separate determinations.

Yeast expression/reporter system using the mouse and human ER The yeast strain BJ2168 (MAT a, prc 1– 407, prb 1–1122, pep 4 –3, leu 2, trip-1, ura 3–52) was transformed with a yeast expression plasmid containing the wild-type mER cDNA, and a reporter plasmid containing a single vitellogenin estrogen responsive element (ERE) linked to the LacZ gene (25). The transformations were performed using the lithium acetate technique, and the yeast transformants were selected by uracil and/or tryptophan auxotrophy. A yeast culture was grown overnight at 30 EC in complete minimal dropout medium lacking uracil and tryptophan. The yeast culture was diluted to OD600 5 0.3 and grown for 2 h before the addition of the environmental compounds alone or in combination (see Fig. 3). The compounds tested in this system were dissolved in dimethyl sulfoxide (DMSO) and 10 ml of an individual compound, or 5 ml each of two compounds were added to 1 ml of yeast culture such that the concentration of DMSO did not exceed 1% by vol. The yeast cultures were grown for an additional 2.5 h before harvesting for measurement of b-galactosidase (b-gal) activity. Yeast cells were pelleted by centrifugation and resuspended in 1 ml Z buffer (60 mm Na2HPO4, 40 mm NaH2PO4, 10 mm KCl, 1 mm MgSO4, 40 mm b-mercaptoethanol, pH 7.0). An aliquot of the suspension was measured for cell density at OD600. A 100-ml aliquot of yeast suspension was then diluted to 1 ml with Z buffer and then treated with 12 ml 0.1% SDS and 15 ml chloroform. The samples were mixed by vortexing and placed at 30 EC for 15 min to permeabilize the cells. Then 200 ml onitrophenyl b-D-galactopyranoside (4 mg/ml in 0.1 m KH2PO4, pH 7.0) were added to the yeast cell suspension and further incubated until chromogenic development. The reactions were stopped by adding 500 ml 1 m Na2CO3 to the samples. Then yeast suspensions were centrifuged

FIG. 1. Competitive binding of organochlorine pesticides to the mouse uterine. Uterine ER was incubated with 1028 M [3H]E2 and different concentrations of unlabeled E2 (d), toxaphene (å), dieldrin (f), and dieldrin/toxaphene (M), and the displacement of radiolabeled hormone was determined as described in Materials and Methods. Only unlabeled E2 significantly decreased binding of [3H]E2 to the uterine ER. to pellet the cellular debris, and the supernatant was analyzed by spectrophotometry at OD420 to quantify b-gal activity. The activity induced by the various chemical treatments were expressed as fold stimulation divided by the vehicle control value determined with DMSO induction. Measurement of b-gal activity after 16 h of chemical treatment was performed in a similar manner, except that the overnight yeast culture was initially diluted to OD600 5 0.1 and then immediately incubated in the presence of the chemical compounds. The yeast strain BJ2407 (a/a prb1–1122/prb1–1122 prc1– 407/prc1– 407 pep4 –3/pep4/3 leu2/leu2 trp1/trp1 ura3–52/ura3–52) was transformed with the ER expression plasmid YePE10, and a reporter vector YRPE2 containing two copies of the vitellogenin ERE was inserted into the promoter of an enhancerless CYC-1-b-gal fusion (26, 27). Individual transformants were grown overnight to a density (OD600) of approximately 0.8 and then combined with an equal volume of minimal medium containing copper sulfate (to induce receptor synthesis from the CUP1 promoter), E2, or the compound to be tested. The final concentration of copper sulfate was 50 mM. Increasing concentrations of ligands dissolved in DMSO were tested as indicated in the figure legends. Cells were induced for 24 h in 96-well plates and then lysed, and b-galactosidase activity was measured. Relative b-gal units were calculated using the following formula: u 5 1000 z [OD420 2 (1.75 z OD550)]/ (0.1 z OD600 z min).

Results

In vitro studies have reported that both dieldrin and toxaphene are weakly estrogenic and exhibit weak binding to the human ER (5, 6, 18). The results summarized in Fig. 1 show that neither dieldrin nor toxaphene (1028–1025 m) significantly bound to the mouse uterine ER in a competitive binding assay using [3H]E2 as the radioligand. In addition, no significant binding was observed using an equimolar concentration of a dieldrin/toxaphene mixture (Fig. 1), and these results contrast with previous studies that reported synergistic binding of this mixture to recombinant baculovirus expressed hER (18). The estrogenic effects of toxaphene, dieldrin, toxaphene plus dieldrin, and E2 were determined in the immature B6C3F1 mouse uterus (Table 1). Treatment with E2 (0.0053 mmol/kgzday) resulted in a 3.1-, 4.8-, and 7.8-fold increase in uterine wet weight, peroxidase activity, and PR

DIELDRIN AND TOXAPHENE ESTROGENIC ACTIVITIES TABLE 1. Effects of E2, dieldrin (D), toxaphene (T), and dieldrin plus toxaphene (D 1 T) in the immature B3C3F1 female mouse uterusa Treatment (dose, mmol/kg)

Control E2 (0.0053) T (2.5) T (15) T (60) T (275) D (2.5) D (15) D (60) D 1 T (2.5) D 1 T (15) D 1 T (60)

Uterine wet weight (mg)

PR (fmol/ute)

UPO (U/ute)

13.6 6 1.6 42.6 6 8.4b 13.4 6 2.4 10.0 6 1.8 14.8 6 1.8 8.1 6 0.8 10.6 6 1.1 12.3 6 1.0 11.1 6 1.4 13.6 6 2.6 13.6 6 3.9 12.9 6 1.5

209 6 113 1619 6 285b 138 6 67 105 6 37 353 6 37 95 6 3 104 6 51 177 6 89 227 6 29 389 6 3b 244 6 46 470 6 35b

0.73 6 0.14 3.50 6 0.12b 1.08 6 0.02 1.10 6 0.14 1.12 6 0.06 0.56 6 0.05 0.61 6 0.02 1.24 6 0.17 0.89 6 0.12 0.84 6 0.06 1.07 6 0.2 0.89 6 0.09

a

Animals were treated with the various compounds for 3 days before termination as described in Materials and Methods. The doses are reported as mmol/kg z day. The uterine wet weight data are means 6 SE from six to nine uteri. PR binding and uterine UPO activity data are means 6 SE from triplicate determinations with extracts from pooled uteri as described under Materials and Methods. A UPO activity unit is defined as the amount of enzyme required to produce an increase of 1 absorbance U/min under the assay conditions. Statistical significance was determined using Dunnett’s onetailed test (ANOVA). b Significantly different from control group (P , 0.01).

binding, respectively. At doses of 2.5, 15, and 60 mmol/ kgzday (33), dieldrin did not significantly induce uterine wet weight or PR binding; however, a slight increase in UPO activity was observed only at the intermediate dose of dieldrin (15 mmol/kg). The highest dose of dieldrin used in this study (275 mmol/kg) was lethal to the animals. Similar results were obtained with the dieldrin/toxaphene mixture (275 mmol/kg). The dose-dependent estrogenic effects of toxaphene also were investigated in the immature mouse uterus (Table 1). No significant increases were observed in uterine wet weight or PR binding at any of the doses (2.5, 15, 60, and 275 mmol/kgzday). UPO activity was increased slightly at the three lower doses, whereas all the uterine responses were decreased at the 275-mmol/kg dose, and this was caused by some toxicity observed in this treatment group. Immature mice also were cotreated with equimolar doses of toxaphene and dieldrin combined (2.5, 15, and 60 mmol/kgzday 33) to investigate possible synergistic interactions between the two weakly estrogenic pesticides, as previously described (18). The results (Table 1) showed that treatment with the binary mixture did not cause a dosedependent increase in uterine wet weight, peroxidase activity, or PR binding; however, PR levels at the 2.5- and 60mmol/kgzday dose were significantly induced, whereas no significant induction was observed with the compounds alone. Previous studies have reported that both toxaphene and dieldrin were weakly estrogenic in MCF-7 human breast cancer cells (5) and their potencies, relative to E2, were 0.0001. The estrogenic activity of toxaphene, dieldrin, and their combination was investigated in this study in transient transfection studies using two estrogen-responsive constructs, namely CKB-CAT and CATH-CAT, which contain 59-promoter sequences from the rat CKB and cathepsin D genes

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TABLE 2. CAT activity in MCF-7 cells treated with E2, dieldrin (D), toxaphene (T), and dieldrin plus toxaphene (D 1 T), and cotransfected with hER and CKB-CAT or CATH-CAT constructsa Treatment (conc.,

M)

Relative % CAT activity CKB-CAT

CATH-CAT

100 6 8.2 2565 6 355

100 6 7.4 384 6 66

D (1028) (1027) (1026) (1025)

98 6 6.3 86 6 8.5 97 6 2.4 93 6 2.7

98 6 11 110 6 18 99 6 1.7 88 6 6.2

T (1028) (1027) (1026) (1025)

101 6 6.9 108 6 5.7 124 6 11 108 6 3.4

101 6 2.6 96 6 3.6 93 6 7.4 99 6 5.0

D 1 T (1028) (1027) (1026) (1025)

95 6 9.6 118 6 13 100 6 13 82 6 4.6

138 6 5.1 125 6 9.3 108 6 6.0 104 6 1.4

DMSO (control) E2 (1029)

a MCF-7 cells were transiently transfected with hER (4 or 5 mg) and CKB-CAT (10 mg) or CATH-CAT (5 mg) constructs and treated with various chemicals. CAT activity was determined as described in Materials and Methods. Results are expressed as means 6 SE for three separate experiments.

FIG. 2. Effects of E2 and organochlorine pesticides on proliferation of MCF-7 cells. The cells were treated with 1029 M E2 (- - - -) or 1027–1025 M dieldrin (L), toxaphene (V), or dieldrin/toxaphene (f) for 11 days, as described in Materials and Methods. The results are presented as means of three separate experiments in which SD , 15%. Significant (*, P , 0.05) induction of cell growth was observed only for 1027 M toxaphene and 1027 M dieldrin/toxaphene mixture; 1029 M E2 caused more than a 6-fold induction in cell proliferation.

linked to a bacterial CAT reporter gene (20, 21). The results summarized in Table 2 indicate that at concentrations ranging from 1028–1025 m, neither toxaphene nor dieldrin induced CAT activity in MCF-7 cells transiently transfected with either the CKB-CAT or CATH-CAT constructs. In contrast, 1029 m E2 induced a 24-fold increase in CAT activity in MCF-7 cells transiently transfected with CKB-CAT and a 3.8-fold increase in cells transiently transfected with the CATH-CAT construct. Combined treatment of MCF-7 cells with an equimolar mixture of dieldrin plus toxaphene (1028– 1025 m) resulted in no induced response and was similar to

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that observed for the individual compounds. The results in Fig. 2 summarize the concentration-dependent effects of toxaphene, dieldrin, and toxaphene plus dieldrin (equimolar concentrations) on proliferation of MCF-7 cells. There was not a concentration-dependent increase in cell proliferation by either compound alone or the mixture (1027–1025 m); however, small but significant increases in cell proliferation were observed for toxaphene and dieldrin plus toxaphene at the 1027 m concentration. In contrast to both organochlorine pesticides, E2 induced a 6-fold increase in MCF-7 cell proliferation. The relative competitive binding of the organochlorine pesticides was determined by incubating MCF-7 cells with 1029 m [3H]E2 in the presence or absence of 2 3 1027 m unlabeled E2 (to determine nonspecific binding), toxaphene (1025 m), dieldrin (1025 m), and equimolar concentrations of the dieldrin plus toxaphene mixture (1025 m). Total cell extracts were obtained, and after treatment with DCC, radioligand-bound ER binding was determined. The results (Table 3) demonstrate that binding of [3H]E2 to the ER in MCF-7 cells was not competitively decreased by cotreatment with 1025 m toxaphene, dieldrin, or the toxaphene/dieldrin mixture, whereas a 200-fold excess of unlabeled E2 caused a 94% decrease in ER binding (the specifically-bound ER was the difference between the total binding minus the binding after competition with unlabeled E2). These results indicate minimal competition by toxaphene, dieldrin, or dieldrin plus toxaphene for the ER in MCF-7 cells using this whole cellbinding assay. Two yeast-based ER assays were used to investigate synergistic interactions of organochlorine pesticides, as previously reported (18). The results in Fig. 3 summarize the effects of toxaphene, dieldrin, and other pesticides on b-gal activity in a yeast system using the mER and a reporter plasmid containing a single ERE linked to the LacZ gene (25). At a concentration of 2.5 3 1027 m, the pesticides did not induce b-gal activity (Fig. 3A), as previously reported using a yeast assay system expressing the hER (18). However, 2.5 3 1025 m of any compound individually was capable of inducing activity from 1.5-fold (dieldrin) to 4-fold (endosulfan). This induction was approximately 7–18% of the activity induced by 1 3 1028 m Diethylstilbestrol (DES), which was 22-fold. No synergistic induction by the mER was demonTABLE 3. Competitive binding of E2, dieldrin (D), toxaphene (T), and dieldrin plus toxaphene (D 1 T) to the ER in MCF-7 cellsa Unlabeled competitor (conc., M)

E2 (2 3 1027) D (1025) T (1025) D 1 T (1025; equimolar)

Binding (DPM) [3H]E2 (1029 M)

[3H]E2 1 unlabeled competitor

5059 6 773 5580 6 217 5603 6 126 5608 6 585

308 6 45 5120 6 436 4620 6 369 5423 6 586

a MCF-7 cells were treated for 2 h with 1029 M [3H]E2 in the presence or absence of various unlabeled competitors; whole cell extracts were isolated, treated with dextran-coated charcoal to remove unbound ligands, and ER binding was determined as described in Materials and Methods. The specific binding (5059 2 308) was 4751 DPM. Significant competition for [3H]E2 binding was observed only for toxaphene (P , 0.05). Results are expressed as means 6 SE for three separate determinations.

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strated with any of the chemical combinations ranging from 1–3 3 1027 m. Furthermore, the chemical mixtures did not induce activity above background, as in the case of each compound individually at 2.5 3 1027 m. Synergistic transactivation also was absent when any two compounds were mixed at the higher concentration (2.5 3 1025 m) (Fig. 3A). The maximum level of activity attained by any chemical

FIG. 3. b-gal activity induced by environmental compounds using an estrogen-responsive reporter system in yeast expressing mouse ER. Yeast cells expressing wild-type mER were exposed to the environmental chemicals chlordane (C), dieldrin (D), toxaphene (T), and endosulfan (E), individually at 2.5 3 1025 M (closed bars). The yeast cells also were exposed to the same chemicals individually at 2.5 3 1027 M (open bars) or in combination at equivalent molar concentrations (see below) reported to produce synergistic induction of halfmaximal b-gal activity (open bars). The final concentrations used for each combination of compounds are: E1D 5 1027 M; E1T 5 1027 M; E1C 5 2 3 1027 M; D1T 5 2 3 1027 M; D1C 5 3 3 1027 M; T1C 5 3 3 1027 M. The yeast cells also were exposed to DES at 1028 M (closed bar) and 1029 M (open bar) as a positive transactivation control. The stimulation of b-gal activity by the compounds is determined as fold stimulation over the background activity induced by DMSO. The yeast cells were exposed to the chemicals for 2.5 h (Fig. 3A) or 16 h (Fig. 3B) before measurement of b-gal activity. Each data bar represents the average and SD of four independent measurements.

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alone or in combination at 2.5 3 1025 m was never above 18% of the level obtained with 1 3 1028 m DES. This observation directly contrasts with the hER system, which showed that binary mixtures of these compounds synergistically induced up to 65% of the activity observed for 1028 m E2 (18). A further comparison of mER with the hER (18) yeast-based systems was determined using a longer (16 h) incubation time (Fig. 3B). Induction by 1028 m DES was increased to 71-fold; however, the organochlorine pesticides alone (2.5 3 1027 m) or in combination (1 to 3 3 1027 m) were inactive. At higher concentrations (2.5 3 1025 m), only dieldrin and endosulfan were weakly estrogenic, and synergism was not observed for the binary mixtures. The estrogenic activity of dieldrin, toxaphene, other organochlorine pesticides, and their binary mixtures also were investigated using yeast transformants containing the human ER plus a reporter gene containing two vitellogenin EREs upstream of the b-gal gene (26, 27). The cells were treated with increasing concentrations of either E2 as a positive control or endosulfan, chlordane, toxaphene, and dieldrin (singly or in combination) (Fig. 4). E2 (1028 m) induced a 5000-fold increase in b-gal activity. Of the other chemicals tested, only endosulfan (1024 m) induced b-gal activity above background. Equimolar concentrations of combinations of endosulfan, chlordane, toxaphene, and dieldrin did not result in synergistic induction of b-gal activity. The failure to observe synergistic interactions for dieldrin plus toxaphene and other binary mixtures in the two yeast-based assays (Figs. 3 and 4) was consistent with results observed in the

FIG. 4. b-gal activity induced by environmental compounds using an estrogen-responsive reporter system in yeast expressing human ER. The experiment was performed as described above, except that yeast strain BJ2407, transformed with a yeast expression plasmid containing the wild-type human ER and a reporter plasmid containing two copies of the vitellogenin ERE linked to the LacZ gene, was used to match experimental conditions reported by Arnold and co-workers (18). The yeast were exposed to environmental chemicals alone or in combination for 24 h in 96-well plates and then assayed for b-gal activity.

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mouse uterus (Fig. 1 and Table 1) and MCF-7 human breast cancer cells (Fig. 2 and Table 2). Discussion

Several recent studies have characterized the estrogenic activities of a diverse spectrum of industrial compounds which includes organochlorine pesticides, PCBs, hydroxy PCBs, phthalates, and various phenolics such as nonylphenol and nonylphenol ethoxylates (4 – 6, 8, 10, 18, 28). Exposure to persistent organochlorine pollutants has been associated with reproductive problems in some wildlife populations, and it has been hypothesized that endocrine-disrupting properties of these environmental contaminants may be responsible for the adverse developmental effects (1). Environmental levels of most organochlorine pesticides have decreased in North America and other developed countries where their applications have been restricted or banned (29, 30). Arnold and co-workers (18) recently reported that interactions between weakly estrogenic pesticides such as dieldrin, toxaphene, and endosulfan resulted in enhanced binding to baculovirus-expressed hER and synergistic induction of estrogen-responsive reporter gene activity in a yeast bioassay. It was surprising that toxaphene alone was only weakly estrogenic because toxaphene is a mixture of C10H10Cl8 isomers that undoubtedly exhibit estrogenic and nonestrogenic activities. Interestingly, chlordane also synergistically interacted with the estrogenic pesticides in the same assay system, even though chlordane does not seem to be estrogenic (6, 18). These results have significant mechanistic implications regarding ER-mediated gene expression; moreover, the apparent synergistic effects observed for the weakly estrogenic and nonestrogenic organochlorine pesticides have heightened concern regarding potential environmental and human health implications of exposure to mixtures of environmental contaminants (31, 32). This study was designed to further investigate the estrogenic activity and interactions of toxaphene and dieldrin using three well-characterized models, namely the immature mouse uterus, MCF-7 human breast cancer cells, and the yeast-based ER assay. The results illustrated in Fig. 1 demonstrate that toxaphene, dieldrin, and a mixture containing equimolar concentrations of both compounds do not competitively displace [3H]E2 from the mouse uterine ER. A previous study (33) also showed that dieldrin (3 mg/kg 37) and toxaphene (25 mg/kg 37) and several other organochlorine pesticides did not induce a uterine wet weight increase in the immature female rat. In this study, using a standard immature mouse protocol for estrogenicity, toxaphene and dieldrin alone exhibited minimal estrogenic activity. The toxaphene/dieldrin mixture induced a small increase in uterine PR levels (Table 1); however, this response was not dose dependent. For the remaining responses, no apparent synergistic interactions were observed for the toxaphene/dieldrin mixture (Table 1). These results indicate that there are major differences between the interactive estrogenic effects and ER binding of toxaphene and dieldrin in the mouse uterus (Table 1; Fig. 1) compared with results obtained with the hER construct used

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in the baculovirus-expressed system or the yeast assay system used by Arnold and co-workers (18), even though the mouse ER binds with variable affinities to other environmental chemicals such as hydroxy-PCBs (3) and alkylphenols (10). The potential interactions of toxaphene and dieldrin were investigated further in MCF-7 human breast cancer cells. Soto and co-workers (5) previously reported that both toxaphene and dieldrin induced proliferation of MCF-7 cells in their E-screen assay; however, this response was only induced at the highest concentration (1025 m). In the present study, 1025 m toxaphene, 1025 m dieldrin, and 1025 m dieldrin plus toxaphene did not induce cell proliferation, whereas 1029 m E2 caused a 6-fold increase in cell growth (Fig. 2). In contrast, other xenoestrogens previously identified as weak estrogens in the E-screen, including nonylphenol, 29,49,69-trichloro-4-biphenylol and bisphenol-A, induced MCF-7 cell proliferation in our assay system (data not shown). The reason for the differences between results of this study with toxaphene and dieldrin vs. previous reports (5, 6) may be caused by several factors, including cell culture conditions, cell passage number, or serum (34). The estrogenic activity of toxaphene, dieldrin, and toxaphene plus dieldrin also was investigated in transient transfection assays, which used two constructs (CKB-CAT and CATH-CAT) that contain estrogen-responsive 59-promoter sequences from the CKB and cathepsin D genes (20, 21). In transient transfection studies, E2 significantly induced CAT activity in MCF-7 cells, whereas induction was not observed with toxaphene, dieldrin, or their combination (Table 2). These results complement ER-binding studies, which showed that the organochlorine pesticides (alone or in combination) did not competitively bind to the hER in MCF-7 cells (Table 3). The effects of toxaphene, dieldrin, other organochlorine pesticides, and binary mixtures of these compounds also were investigated in yeast-based reporter gene assays (25– 27). The results obtained in yeast transformed with an expression plasmid that contained the wild-type mouse ER and a reporter plasmid containing a single ERE linked to the LacZ gene (Fig. 3) indicated that the estrogenic activities of dieldrin and toxaphene were minimal, and no synergistic effects were observed for a binary mixture of these and other organochlorine pesticides. The potential interactions of toxaphene and dieldrin, as well as endosulfan and chlordane, also were investigated in a yeast-based ER assay that used the same yeast strain and reporter gene construct as reported by Arnold and co-workers (18). In contrast to the previous study, synergistic activity was not observed for binary mixtures of toxaphene/dieldrin or other pesticide combinations. Endosulfan exhibited estrogenic activity in both yeast assays; however, binary mixtures of endosulfan plus the other organochlorine compounds did not induce synergistic responses (Figs. 3 and 4). The reasons for the differences between results of this study and that reported by Arnold and co-workers (18) are unclear but may be caused by a unique aspect of the yeast strain maintained in either laboratory or to differences in ER constructs used by either laboratory. The differences could not be accounted for by levels of ER expression because varying the level of ER expression did not alter the results (data not shown). These data (Fig. 4) dem-

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onstrate that synergism between weakly estrogenic chemicals is not universal, even within the same strain of yeast. In summary, this study has compared the estrogenic activities of toxaphene, dieldrin, and an equimolar mixture of the two compounds in both ER-binding and functional assays in the mouse uterus, MCF-7 human breast cancer cells, and in estrogen-responsive yeast assays. The activities of both compounds alone were minimal, and no synergistic effects were observed with the binary mixture in the mouse uterus and MCF-7 cells (Tables 1–3; Figs. 1 and 2). Similar results were observed for toxaphene/dieldrin and other binary mixtures of organochlorine insecticides in two yeastbased assays (Figs. 3 and 4). These data contrasted with results reported by Arnold and co-workers (18) in the ERbinding and yeast assays and other studies which have reported nonadditive (synergistic) interactions with hydroxy PCBs and other mixtures of organochlorine pesticides and phenolics (6, 10). It should be noted also that results of previous studies with mixtures of estrogenic/nonestrogenic compounds do not always give synergistic responses. Toxaphene is not a single chemical but a chlorinated camphene mixture containing several C10H10Cl8 isomers that undoubtedly exhibit estrogenic and nonestrogenic activity. However, the toxaphene mixture is only weakly estrogenic or nonestrogenic in both in vitro and in vivo assays (6, 18, 33). Similarly, Aroclor 1221 and nonylphenol also are mixtures of active and inactive congeners, and the estrogenic activities of both mixtures also are relatively weak (5–7, 10, 35). This suggests that synergistic interactions of weakly estrogenic or nonestrogenic chemicals may be highly structure dependent. Moreover, the results obtained for a binary mixture of dieldrin and toxaphene and other organochlorine pesticides in this study indicate that synergistic interactions are both response- and assay-specific. Thus, the recent scientific, regulatory, and public concern regarding the potential adverse environmental and human health impacts associated with the synergistic effects induced by organochlorine pesticide mixtures (18) should be tempered by the results reported in this study, which show that in 10 different assays, synergistic interactions were not observed (36). References 1. Colborn T, Vom Saal FS, Soto AM 1993 Developmental effects of endocrinedisrupting chemicals in wildlife and humans. Environ Health Perspect 101:378 –384 2. Birnbaum L 1995 Developmental effects of dioxin. Environ Health Perspect 103:89 –94 3. Korach KS, Sarver P, Chae K, McLachlan JA, McKinney JD 1988 Estrogen receptor-binding activity of polychlorinated hydroxybiphenyls: conformationally restricted structural probes. Mol Pharmacol 33:120 –126 4. Soto AM, Justicia H, Wray JW, Sonnenschein C 1991 p-Nonylphenol: an estrogenic xenobiotic released from “modified” polystyrene. Environ Health Perspect 92:167–173 5. Soto AM, Chung KL, Sonnenschein C 1994 The pesticides endosulfan, toxaphene, and dieldrin have estrogenic effects on human estrogen-sensitive cells. Environ Health Perspect 102:380 –383 6. Soto AM, Sonnenschein C, Chung KL, Fernandez MF, Olea N, Serrano FO 1995 The E-screen assay as a tool to identify estrogens - an update on estrogenic environmental pollutants. Environ Health Perspect 103:113–122 7. Krishnan AV, Stathis P, Permuth SF, Tokes L, Feldman D 1993 Bisphenol-A: an estrogenic substance is released from polycarbonate flasks during autoclaving. Endocrinology 132:2279 –2286 8. White R, Jobling S, Hoare SA, Sumpter JP, Parker MG 1993 Environmentally persistent alkylphenolic compounds are estrogenic. Endocrinology 135:175–182 9. Jobling S, Reynolds T, White R, Parker MG, Sumpter JP 1995 A variety of

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