2000 Progress Report: Species-Specific Endocrine Disruption: PCB- and PAH-Induced Estrogenic EffectsEPA Grant Number: R826301
Title: Species-Specific Endocrine Disruption: PCB- and PAH-Induced Estrogenic Effects
Investigators: Zacharewski, Timothy
Institution: Michigan State University
EPA Project Officer: Carleton, James N
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: Economics and Decision Sciences , Endocrine Disruptors , Health , Safer Chemicals
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.
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|
||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.||