Grantee Research Project Results
2000 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, 1999 through December 31, 2000
Project Amount: $282,998
RFA: Endocrine Disruptors (1997) RFA Text | Recipients Lists
Research Category: Environmental Justice , Endocrine Disruptors , Human Health , Safer Chemicals
Objective:
The objectives of this research project are to examine the alleged estrogen receptor-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 our general hypothesis that 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 estrogen receptor (ER) ligand binding domains.
Progress Summary:
Species Comparison of Gal4-ERdef-Mediated Transactivation. This study examined
the ability of 15 natural and synthetic estrogenic chemicals to induce gene
expression mediation through both ER subtypes and ERs different species. MCF-7
cells were transiently co-transfected with a Gal4-ER chimeric receptors consisting
of the D, E, and F domains of either the human alpha (Gal4-hER), mouse alpha
(Gal4-mER
), mouse beta (Gal4-mER
), chicken (Gal4-cER), green anole (Gal4-
ER),
xenopus (Gal4-xER), or rainbow trout ERs (Gal4-rtERdef), and a Gal4 regulated
luciferase reporter gene. The ability of 17
-estradiol (E2) to induce reporter
gene expression was similar for 6 of the 7 chimeric receptors, with EC50 values
ranging from 0.05 to 0.7 nM. The ability of E2 to induce reporter gene expression
mediated by Gal4-rtER was 2 orders of magnitude lower, with an EC50 value of
28 nM at 37ºC. This discrepancy was primarily due to temperature, because
at 20ºC, only 9-fold differences in EC50 values were observed. Therefore,
the ability of the estrogenic chemicals to induce Gal4-rtER mediate gene expression
was assayed at 37ºC and 20ºC. In general, the ability of several compounds
to induced Gal4-rtER mediated gene expression increased at 20ºC. Although
the response of E2 was similar among the ERs, many differences were observed.
For example,
-zearalenol induced reporter gene expression mediated by Gal4-rtER
at lower concentrations than E2, which was in contrast to other Gal4-ERs. Coumestrol-induced
reported gene expression 280- and 14-fold greater mediated through Gal4-mERb
and Gal4-aER, respectively, than through the other Gal4 chimeric receptors.
These data show that certain estrogenic compounds exhibit a differential ability
to induce reporter gene activity mediated by both ER subtypes and ERs from different
species. Also, it illustrates the importance of temperature when examining rtER
mediated activity.
Preferential Interaction of PAH-Related Compounds With Er. We examined
the ability of several 4- and 5-ring polycyclic aromatic hydrocarbon (PAH)-related
compounds to interact with the ER alpha and beta isoforms. The compounds, many
of which had been previously studied for mutagenic potential, consisted of carbazoles
(benzo[a]carbazole and [c]carbazole), benzonaphthothiophenes (benzo[b]naphtho[2,1-d]thiophene
and -[2,3-d]thiophene), and several hydroxylated PAHs and thiophenes (2-OH-,
2-OH-5-methyl-, and 8-OH-5-methyl-chrysene; 2-OH-benzo[c]phenanthrene; 3-OH-benzo[b]naphtho[2,1-d]thiophene;
and 3-OH-benzo[b]phenanthro[2,3-d]thiophene). We performed receptor binding
affinity investigations by assessing the compounds' ability to compete with
3H-labeled 17
-estradiol (E2) for binding to either the D, E, and F domains
of human ERa linked to glutathione-S-transferase (GST-hERadef) or to full-length
human ERb. The receptor-ligand complex's ability to transactivate ER-regulated
genes was assessed using MCF-7 cells transiently transfected with either a Gal4-human
ERadef or Gal4-mouse ERbdef construct, as well as a Gal4-regulated reporter
construct. Only those compounds containing a hydroxyl group showed significant
binding, which was comparable for both isoforms (IC50 range approximately 20-300
nM; E2 IC50 approximately 3 nM). However, nearly all compounds were able to
induce reporter gene expression preferentially through mERb. In fact, only in
the mERb system were most compounds able to achieve maximal levels (60-100 percent
of those obtained with E2) at 10 mM, with EC50 values ranging from 30-600 nM
(E2 EC50 approximately 200 pM). These data support previous evidence, suggesting
that even while some compounds may possess a similar affinity for both ER isoforms,
the capacity for transcriptional activation can still be isoform-specific.
Establishment of Xenopus laevis as a Model for Investigating In
Vitro and In Vivo Endocrine Disruption in Amphibians. In this
study, in vitro ER competitive binding and gene expression assays, and
the induction of vitellogenin mRNA in vivo, were used to assess Xenopus
laevis as an amphibian model for examining potential endocrine disruptors.
Competitive binding to Xenopus ER was investigated using a bacterially
expressed fusion protein consisting of glutathione-S-transferase (GST) linked
to the ER ligand binding domain (LBD) of Xenopus (GST-xER). Equilibrium
analysis revealed that the saturation was reached at a concentration of 10 nM
[3H]17-estradiol
(E2) with a dissociation constant (Kd) of 6.4 ±
1.3 nM. In a competitive binding assay, the IC50 value
for E2 was 12.2 ± 0.34 nM. The ability of E2 to induce Xenopus
ER-regulated gene expression was assessed in MCF-7 human breast cancer cells
transiently transfected with a chimeric receptor consisting of the Gal4 DNA
binding domain linked to the Xenopus ER LBD (Gal4-xER) and a Gal4-regulated
luciferase reporter gene, 17m5-G-Luc. Treatment of E2 resulted in approximately
30-50 fold maximal induction of reporter gene activity with an EC50
of 0.67 + 0.31 nM. Adult male Xenopus were intraperitoneally treated
for 3 consecutive days with E2 at 0, 0.05, 0.1, 0.5, 1, and 5 mg/kg for a period
of 12 days. The level of vitellogenin induction was determined using semiquantitative
RT-PCR. E2 treatment showed a dose response increase in vitellogenin mRNA with
the maximal induction at an accumulative dose of 3 mg/kg E2 (p <0.001, n
= 35). In summary, we have developed three complementary methods to detect estrogenic
effects of E2 using Xenopus as a model. These methods can be used to
assess the estrogenicity of a substance in Xenopus as a model amphibian species.
Journal Articles on this Report : 1 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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Celius T, Matthews JB, Giesy JP, Zacharewski TR. Quantification of rainbow trout (Oncorhynchus mykiss) zona radiata and vitellogenin mRNA levels using real-time PCR after in vivo treatment with estradiol-17 beta or alpha-zearalenol. Journal of Steroid Biochemistry and Molecular Biology 2000;75(2-3):109-119. |
R826301 (2000) R826301 (Final) |
not available |
Supplemental Keywords:
endocrine, endocrine receptor, polychlorinated biphenyl, PCB, polyaromatic hydrocarbon, PAH, fish, frog, mouse, bird., RFA, Health, Scientific Discipline, Toxics, Waste, Environmental Chemistry, Health Risk Assessment, HAPS, Endocrine Disruptors - Environmental Exposure & Risk, chemical mixtures, endocrine disruptors, 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 effectsProgress 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.