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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 Synergism¹

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

Address all correspondence and requests for reprints to: Stephen Safe, Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466.

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 17ß-estradiol (E₂) (0.0053 kg/day x3) 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 µmol/kg (x3) doses of toxaphene, dieldrin, or dieldrin/toxaphene (equimolar) did not significantly induce a dose-dependent 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 5'-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 (10^-8–10^-5 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 10^-9 M [³H]E₂ in the presence or absence of 2 x 10^-7 M unlabeled E₂ (to determine nonspecific binding), toxaphene (10^-5 M), dieldrin (10^-5 M), and equimolar concentrations of the dieldrin plus toxaphene mixture (10^-5 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 ß-galactosidase (ß-gal) activity in yeast transformed with the human ER and a double estrogen responsive element upstream of the ß-gal reporter gene. Treatment with 10^-6–10^-4 M chlordane, dieldrin, toxaphene, or an equimolar mixture of dieldrin/toxaphene did not induce activity, whereas 10^-4 M endosulfan caused a 2000-fold increase in ß-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 ß-gal reporter gene. Dieldrin, chlordane, toxaphene, and endosulfan induced a 1.5- to 4-fold increase in activity at a concentration of 2.5 x 10^-5 M. Synergistic transactivation was not observed for any equimolar binary mixture of the pesticides at concentrations of either 2.5 x 10^-5 M or 2.5 x 10^-4 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.

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