2001 Progress Report: Species-Specific Endocrine Disruption: PCB- and PAH-Induced Estrogenic Effects

EPA Grant Number: R826301
Title: Species-Specific Endocrine Disruption: PCB- and PAH-Induced Estrogenic Effects
Investigators: Zacharewski, Timothy
Institution: Michigan State University
EPA Project Officer: Turner, Vivian
Project Period: January 1, 1998 through December 31, 2000
Project Period Covered by this Report: January 1, 2000 through December 31, 2001
Project Amount: $282,998
RFA: Endocrine Disruptors (1997) RFA Text |  Recipients Lists
Research Category: Economics and Decision Sciences , Endocrine Disruptors , Health , Safer Chemicals


The objective of this research project is to examine the alleged estrogen receptor (ER)-mediated activities of selected environmentally relevant compounds using a combination of in vitro and in vivo assays. These studies are being performed in a number of species including fish, frogs, mice, and birds, to test the following general hypothesis: rodents are not appropriate surrogates for identifying and assessing the risks of alleged environmental estrogens to human and wildlife health due to a lack of significant amino acid sequence homology between species in the ER ligand binding domains.

Progress Summary:

Identification of Residues within Human ERa and Rainbow Trout ER that Contribute to Temperature Sensitivity and Interaction with Estrogenic Compounds

The human estrogen receptor-alpha (hER) and the rainbow trout ER (rtER) have different functional activities in response to E2 and other estrogenic chemicals. Amino acid residues that differ between hER and rtER ligand binding pockets were identified; L349 and M528 in hER, and M317 and I496 in rtER. Reciprocal mutants, hER (L349M, M528I) and rtER (M317L, I496M), were prepared. The affinity (Kd value) of GST-ER fusion proteins consisting of the D, E, and F domains of hER (GST-hER) and rtER (GST-rtER) for E2 was 0.5 nM and 1.0 nM at 4ºC, respectively. Although the GST-hER Kd value was unchanged at 20ºC, 30ºC, and 37ºC; that for GST-rtER was 1.8-, 4.4-, and 5.2-fold higher at 20ºC, 30ºC, and 37ºC, respectively. The Kd value for the M317L;I496M mutant of GST-rtER was 0.85-, 1.5-, and 1.8- fold different compared to wild type. The ability of E2 to induce reporter gene activity in transiently transfected MCF-7 cells maintained at 37ºC was 280-fold greater for Gal4-hER than for Gal4-rtER, with EC50 values of 0.01 nM and 28 nM, respectively. However, when MCF-7 cells were maintained at 30ºC and 20ºC, only a 30- and 9-fold difference was observed, respectively. The L349M;M528I mutant of Gal4-hER caused a 20-fold reduction in E2-induced reporter gene activity at 37ºC, which was reduced 3- and 4-fold at 30ºC and 20ºC, respectively. The E2-induced reporter gene activity mediated by the M315L;I496M mutant of Gal4-rtER was reduced 60-fold when compared to that of Gal4-hER at 37ºC. This effect was reduced to 8- and 3-fold at 30ºC and 20ºC, respectively. In addition, for some chemicals, the double mutants adopted the binding preference and ability to activate gene expression of the other wild type receptor. This suggests that residues L349/M317 and M528/I496 play important roles in the observed differences in ligand binding between the hER and rtER, and in influencing the reduction in E2-induced reporter gene activity and the temperature sensitivity of the rtER.

Future Activities:

Future activities will include further investigation into the molecular basis for species differences in ligand preference and ligand binding affinity. These studies will involve examining the crystal structure of the estrogen receptor occupied by structurally diverse endocrine disruptors to determine how ligand structure affects estrogen receptor protein structure. In addition, the effect of ligand structure on coregulator recruit recruitment and subsequent global gene expression patterns also will be investigated, thus providing mechanistic information that can be used to predict species and tissue specific effects of estrogenic endocrine disruptors.

The establishment of global gene expression profile assays to screen substances for endocrine disrupting activities using microarrays also will be investigated.

Journal Articles on this Report : 5 Displayed | Download in RIS Format

Other project views: All 50 publications 11 publications in selected types All 10 journal articles
Type Citation Project Document Sources
Journal Article Fertuck KC, Matthews JB, Zacharewski TR. Hydroxylated benzo[a]pyrene metabolites are responsible for in vitro estrogen receptor-mediated gene expression induced by benzo[a]pyrene, but do not elicit uterotrophic effects in vivo. Toxicological Sciences 2001;59(2):231-240. R826301 (2001)
R826301 (Final)
not available
Journal Article Fertuck KC, Kumar S, Sikka HC, Matthews JB, Zacharewski TR. Interaction of PAH-related compounds with the and isoforms of estrogen receptor. Toxicology Letters 2001;121(3):167-177. R826301 (2001)
R826301 (Final)
R826192 (Final)
not available
Journal Article Matthews JB, Clemons JH, Zacharewski TR. Reciprocal mutagenesis between human α(L349, M528) and rainbow trout (M317, I496) estrogen receptor residues demonstrates their importance in ligand binding and gene expression at different temperatures. Molecular and Cellular Endocrinology 2001;183(1-2):127-139 R826301 (2001)
R826301 (Final)
not available
Journal Article Matthews J, Celius T, Halgren R, Zacharewski T. Differential estrogen receptor binding of estrogenic substances: a species comparison. Journal of Steroid Biochemistry and Molecular Biology 2000;74(4):223-234. R826301 (2001)
not available
Journal Article Rosen G, O'Bryant E, Matthews J, Zacharewski T, Wade J. Distribution of androgen receptor mRNA expression and immunoreactivity in the brain of the green anole lizard. Journal of Neuroendocrinology 2002;14(1):19-28. R826301 (2001)
R826301 (Final)
not available

Supplemental Keywords:

human health, metabolism, carcinogen, animal, mammalian, organism, cellular, diet, effluent, terrestrial, aquatic, estrogenic endocrine modulators., RFA, Health, Scientific Discipline, Toxics, Waste, Environmental Chemistry, Health Risk Assessment, Endocrine Disruptors - Environmental Exposure & Risk, HAPS, endocrine disruptors, chemical mixtures, Risk Assessments, Biochemistry, Children's Health, Endocrine Disruptors - Human Health, adverse outcomes, complex mixtures, endocrine disrupting chemicals, PCBs, fertility, industrial chemicals, PAH, animal models, carcinogens, human growth and development, cancer, human exposure, estrogen response, reproductive processes, estrogen receptors, biological effects

Relevant Websites:

http://www.bch.msu.edu/~zacharet/ Exit

Progress and Final Reports:

Original Abstract
1999 Progress Report
2000 Progress Report
Final Report