Steven F. Arnold

Certain chemicals in the environment are estrogenic. The low potencies of these compounds, when studied singly, suggest that they may have little effect on biological systems. The estrogenic potencies of combinations of such chemicals were screened in a simple yeast estrogen system (YES) containing human estrogen receptor (hER). Combinations of two weak environmental estrogens, such as dieldrin, endosulfan, or toxaphene, were 1000 times as potent in hER-mediated transactivation as any chemical alone. Hydroxylated polychlorinated biphenyls shown previously to synergistically alter sexual development in turtles also synergized in the YES. The synergistic interaction of chemical mixtures with the estrogen receptor may have profound environmental implications. These results may represent a previously uncharacterized level of regulation of estrogen-associated responses.

The aim of this study was to compare results obtained by eight different short-term assays of estrogenlike actions of chemicals conducted in 10 different laboratories in five countries. Twenty chemicals were selected to represent direct-acting estrogens, compounds with estrogenic metabolites, estrogenic antagonists, and a known cytotoxic agent. Also included in the test panel were 17beta++-estradiol as a positive control and ethanol as solvent control. The test compounds were coded before distribution. Test methods included direct binding to the estrogen receptor (ER), proliferation of MCF-7 cells, transient reporter gene expression in MCF-7 cells, reporter gene expression in yeast strains stably transfected with the human ER and an estrogen-responsive reporter gene, and vitellogenin production in juvenile rainbow trout. 17beta-Estradiol, 17alpha-ethynyl estradiol, and diethylstilbestrol induced a strong estrogenic response in all test systems. Colchicine caused cytotoxicity only. Bisphenol A induced an estrogenic response in all assays. The results obtained for the remaining test compounds--tamoxifen, ICI 182.780, testosterone, bisphenol A dimethacrylate, 4-n-octylphenol, 4-n-nonylphenol, nonylphenol dodecylethoxylate, butylbenzylphthalate, dibutylphthalate, methoxychlor, o,p'-DDT, p,p'-DDE, endosulfan, chlomequat chloride, and ethanol--varied among the assays. The results demonstrate that careful standardization is necessary to obtain a reasonable degree of reproducibility. Also, similar methods vary in their sensitivity to estrogenic compounds. Thus, short-term tests are useful for screening purposes, but the methods must be further validated by additional interlaboratory and interassay comparisons to document the reliability of the methods.

Reports of reproductive abnormalities in the American alligator from Lake Apopka, Florida, have been linked to a spill of DDT and other pesticides suspected of having hormonelike activity. To determine whether environmental chemicals had the potential to function as exogenous hormones in the American alligator, we examined the ability of chemicals to bind the estrogen receptor (aER) and progesterone receptor (aPR) in a protein extract prepared from the oviduct of the alligator. In competition binding assays with [3H]17 beta-estradiol, some DDT metabolites showed inhibition of [3H]17 beta-estradiol binding to aER. A combination of DDTs demonstrated an additive decrease in [3H]17 beta-estradiol binding to aER. Modern-use chemicals such as alachlor, trans-nonachlor, endosulfan, and atrazine also competed with [3H]17 beta-estradiol for binding to the aER. To test the effect of chemicals identified in alligator eggs from Lake Apopka on [3H]17 beta-estradiol binding, we mixed these chemicals at concentrations measured in eggs in the competition binding assay. 2,2-bis(4-chlorophenyl)-N-(methoxymethyl)acetamide (p,p'-DDD) and trans-nonachlor, both found in Lake Apopka, interacted with aER, whereas others such as chlordane and toxaphene did not. Surprisingly, combinations of these chemicals decreased [3H]17 beta-estradiol binding in a greater than additive manner. To assess the ability of chemicals to interact with aPR, we performed commpetition binding assays with the synthetic progestin [3H]R5020. Most of the chemicals tested did not reduce [3H]R5020 binding to aPR, whereas endosulfan, alachlor, and kepone inhibited binding. These results provide the first evidence that environmental chemicals bind the aER and aPR from the American alligator, supporting the hypothesis that the reported reproductive abnormalities may be related to the modulation of endocrine-related responses. The findings that combinations of chemicals demonstrated a greater than additive interaction with the aER and some chemicals bind to the aPR in the competition binding assay are novel. This suggests that interactions of these chemicals with the endocrine system are complex.