Grantee Research Project Results

1998 Progress Report: Methoxychlor and Environmental "Estrogen" Receptors in ER-Minus Mice

EPA Grant Number: R825295
Title: Methoxychlor and Environmental "Estrogen" Receptors in ER-Minus Mice
Investigators: Lubahn, Dennis B.
Current Investigators: Lubahn, Dennis B. , Welshons, Wade V.
Institution: University of Missouri - Columbia
EPA Project Officer: Aja, Hayley
Project Period: February 10, 1997 through February 9, 1999
Project Period Covered by this Report: February 10, 1997 through February 9, 1998
Project Amount: $190,000
RFA: Endocrine Disruptors (1996) RFA Text |  Recipients Lists
Research Category: Endocrine Disruptors , Environmental Justice , Human Health , Safer Chemicals

Objective:

To provide evidence for and clone the putative "methoxychlor" receptor.

Progress Summary:

Effects of estrogens in ERa KO mice
Experiments have been performed to verify the initial in situ hybridization results of estrogen responsiveness in ERa-KO mice with methoxychlor by quantitative RT-PCR (Copy of poster is attached and discussed briefly below) and 4-hydroxy estradiol (PNAS 94:12786-91, 1997; attached) in the ERaKO mice. We have developed a competitive RT-PCR protocol for confirming the quantity of glucose 6-phosphate dehydrogenase and lactoferrin mRNA induction by environmental estrogens in the uterus. These have confirmed our earlier results that there is a novel estrogen response mechanism, which is not ERa OR ERb present in the ERaKO mice.

Technical difficulties because of limited tissue quantities particularly when we begin to characterize unique mRNA responses have been dealt with as we have gained expertise. However, we have continued parallel experiments in our estrogen-treated animals on alternative tissues that may be responsive, in particular liver, which is present in relatively larger quantities and is known to respond to estradiol. We examined lactoferrin, malic enzyme and glucose-6-phosphate dehydrogenase mRNAs responsiveness to estrogens in the liver in ERa-KO mice without much success. We hope that our encouraging efforts with differential display will provide better liver markers. Our problems with limited amounts of uterine tissue to examine methoxychlor responses would then be solved.

Methoxychlor stimulation of lactoferrin and glucose 6 phosphate dehydrogenase mRNAs in ERa-KO Mice
In our original proposal we only presented lactoferrin mRNA induction by methoxychlor at a single time point and concentration with a single technique, in situ hybridization. The results presented below and in the poster (attached) provide strong indications that methoxychlor is likely to be acting through its own putative receptor, which is distinct from ER-a and also the recently discovered ER-b.

Adult wild type (+/+) or homozygous (-/-) ERKO sibling mice of the same mixed genetic background (129/C57BL/6J) were ovariectomized and rested for two weeks before treatment. All treatments were given as two injections of 6 hrs apart of oil (vehicle control), Estradiol-17b (10 mg/kg) and Methoxychlor (3.75, 7.5, 15, 30 and 60 mg/kg) and animals were sacrificed 12 hrs after the final injection. In a separate group of mice Methoxychlor (15 mg/kg was injected together with Estradiol-17b (10 mg/kg). All of the test agents were dissolved in olive oil and injected (0.1 ml/mouse) subcutaneously. A time course study (2, 6, 12, 18, 24 hrs for Methoxychlor and 12, 18 and 24 hrs for estradiol) was done with a single injection of Methoxychlor (15 mg/kg) or estradiol (10 mg/kg). Competitive RT-PCR Assays for lactoferrin and glucose-6-phosphate dehydrogenase mRNAs were developed

Results

One page printout of poster presented at 1998 Endocrine Meeting in New Orleans. Reference is below.

Conclusions

1. The classical estrogen estradiol-17b induced lactoferrin and glucose-6-phosphate dehydrogenase RNA level only in wild type mice not in ER-a knockout type. In contrast the responses of methoxychlor in ER-a knockout mice reveal that its action is not being mediated solely through ER-a.
2. The dose and time dependent response to methoxychlor supports its action through receptor binding and saturation. A clear biphasic nature of response was observed both in wild type and ER-a knockout mice suggesting more than one response mechanism in the ER?-KO mice, i. e. ER-beta AND "methoxychlor" receptors. The differences in the shapes of the curves imply that these "receptors" may be acting differently in their activation of the lactoferrin and glucose-6-phosphate DH genes.
3. Glucose-6-phosphate dehydrogenase is an early responsive gene. In the time course study in the wild type and ER-a knockout mice an early response to estradiol-17b in wild type mice was observed and this pattern of response was also seen in the methoxychlor response also.

Future Activities:

Effects of estrogens on ERb in ERa KO mice
We continue to be intrigued by the potential of ERb to be involved in the methoxychlor response and have begun initial attempt to express human ER-beta and ER-alpha together to see if together they will bind and respond to methoxychlor better. However, in general ERa appears so far to be very much like ERb in its ligand-binding domain specificity. This whole story has become even more complicated with the report by a Pfizer group [Endocrinology 139:1082-1092 (1998)] that there exist alternately spliced forms of ERb. These alternately spliced forms contain an 18 amino acid insert located in the ligand-binding domain, resulting in about 10 fold lower binding affinity for 17b-estradiol. Data were presented comparing the binding affinities of these splice variants to other estrogens, including methoxychlor. All these variants also bound DES and estradiol thus ruling out our novel 'methoxychlor" receptor. Additionally several groups have reported ER-beta and ER-alpha can form heterodimers. No steroid-binding characterization has yet been reported for any of the 12 possible heterodimers. Combined, these two new reports raise the testable possibility of altered ligand specificities with these heterodimers. The possibility remains that the responses we are observing with methoxychlor in ER-a KO mice could be mediated by novel combinations and variants of known ER genes.

In our system, because of the lack of response with 17b-estradiol and competition studies of responsive estrogens with ICI-182, 780 and 17b-estradiol, it is clear to us that the methoxychlor responses we are seeing in ERKO mice are not being mediated by a ligand-binding domain of either ER-a or -b. This supports the existence of a unique non- ER-a or -b receptor, "ER-gamma", IF together as heterodimers they do not possess an altered specificity. A ligand specific for ER-a or -b is still not available either an agonist or an antagonist would be very useful. If it were available it might be possible to design an experiment to distinguish them and then confirm an "ER-gamma" using in vitro and in vivo non-ER-a KO models. We have learned that Karobio, a company in Sweden, has such a compound and we are still attempting to obtain it.

In any case it is likely that ERb will mediate some methoxychlor responses. We are arranging with two labs currently making ERb KO mice that when they become available we will breed a double ER-a and ER-b KO mouse. In this way we will have a cleaner system to determine if there are additional ERs capable of mediating methoxychlor and other estrogens' actions.

Our efforts to express ER-beta were interrupted when it became apparent that all published ER-beta sequences lacked 46 amino acids on their N-terminal ends because of a sequencing error in the original rat ER-beta paper. We have cloned this region in mice and humans and are putting it together with the rest of the mouse and human ER-beta. Efforts to characterize several new additional alternate splice forms of ER-beta found in cows are continuing in work related, but not supported by this grant. We continue to screen mouse genomic DNA with various fish primers to pull out the mammalian homolog. Two new fish cDNAs have been found in unpublished work in two other labs. We are attempting to get these sequences. Differential display efforts are finally beginning to allow us to screen for novel "methoxychlor" receptors. All in all progress is being made, but while we now believe there is firm evidence for a "methoxychlor" receptor we have not yet cloned it.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Publications Views

Other project views:	All 5 publications	2 publications in selected types	All 2 journal articles

Publications

Type	Citation	Project	Document Sources
Journal Article	Das SK, Taylor JA, Korach KS, Paria BC, Dey SK, Lubahn DB. Estrogenic responses in estrogen receptor-α deficient mice reveal a distinct estrogen signaling pathway. Proceedings of the National Academy of Sciences of the United States of America 1997;94(24):12786-12791	R825295 (1998)	not available

Supplemental Keywords:

Methoxychlor Receptor, ER-gamma, RFA, Health, Scientific Discipline, Health Risk Assessment, Environmental Chemistry, Endocrine Disruptors - Environmental Exposure & Risk, Risk Assessments, endocrine disruptors, Children's Health, Molecular Biology/Genetics, Biology, Endocrine Disruptors - Human Health, adverse outcomes, wildlife, molecular mechanisms, endocrine disrupting chemicals, Methoxychlor, receptor bindings, human exposure, animal models, mice, estrogen response, biochemistry, biological effects

Progress and Final Reports:

Original Abstract

1997

Final Report