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.
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 17beta-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 micromol/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 beta-galactosidase (beta-gal) activity in yeast transformed with the human ER and a double estrogen responsive element upstream of the beta-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 beta-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 beta-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.
Ramamoorthy
K
K
Veterinary Physiology and Pharmacology, Texas A&M University, College Station 77843-4466, USA.
Wang
F
F
Chen
I C
IC
Norris
J D
JD
McDonnell
D P
DP
Leonard
L S
LS
Gaido
K W
KW
Bocchinfuso
W P
WP
Korach
K S
KS
Safe
S
S
D000818:Animals; D001667:Binding, Competitive; D001943:Breast Neoplasms / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D004026:Dieldrin / Q000008:administration & dosage / Q000494:pharmacology*; D004357:Drug Synergism; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D006801:Humans; D051379:Mice; D009195:Peroxidase / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D051381:Rats; D011960:Receptors, Estrogen / Q000032:analysis*; D014112:Toxaphene / Q000008:administration & dosage / Q000494:pharmacology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014599:Uterus / Q000187:drug effects* / Q000378:metabolism
D011960:Receptors, Estrogen; D011374:Progesterone; D004958:Estradiol; D014112:Toxaphene; D009195:Peroxidase; D004026:Dieldrin