Grantee Research Project Results
2001 Progress Report: Species-Specific Endocrine Disruption: PCB- and PAH-Induced Estrogenic Effects
EPA Grant Number: R826301Title: Species-Specific Endocrine Disruption: PCB- and PAH-Induced Estrogenic Effects
Investigators: Zacharewski, Timothy
Institution: Michigan State University
EPA Project Officer: Aja, Hayley
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: Environmental Justice , Endocrine Disruptors , Human Health , Safer Chemicals
Objective:
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 |
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| Type | Citation | ||
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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 |
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Fertuck KC, Kumar S, Sikka HC, Matthews JB, Zacharewski TR. Interaction of PAH-related compounds with the α and β isoforms of estrogen receptor. Toxicology Letters 2001;121(3):167-177. |
R826301 (2001) R826301 (Final) R826192 (Final) |
not available |
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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 |
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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 |
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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 effectsRelevant Websites:
http://www.bch.msu.edu/~zacharet/ Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.