**Methoxychlor and Environmental \"Estrogen\" Receptors in ER-Minus
Mice**

# Project Scope {#project-scope .LRI-Heading-2}

Methoxychlor (Mxc) is a pesticide in current use that was developed as a
replacement for dichloro-diphenyltrichloroethane (DDT). Data from
studies in rats indicate that Mxc exposure can elicit physiological
responses similar to estradiol-17β, such as uterine growth and
hypertrophy, increases in uterine peroxidase and ornithine decarboxylase
activities, accelerated vaginal opening, induce persistent vaginal
cornification, and increase uterine estrogen receptor (ER) expression.

The estrogen receptors ERα and ERβ are ligand-activated transcription
factors that form heterodimers or homodimers to regulate estrogen
response gene expression.

The main objectives of this research program were to characterize the
effects of Mxc on estrogen responsive uterine genes and to identify the
putative \"methoxychlor receptor". To address the first objective the
researchers measured lactoferrin (LF) and glucose-6-phosphate
dehydrogenase (G6PD) RNA levels in wild type (WT) and ER-knockout mice
in response to Mxc alone and Mxc in the presence of known ERα ligands.
To address the second objective the researchers took three parallel
approaches: (1) screened a ERα-knockout mouse uterine cDNA library with
probes of the steroid receptor super family via low stringency
hybridization, (2) checked previously cloned "candidate" receptors for
binding to Mxc and its metabolites, (3) expression cloning using
\[3H\]-methoxychlor.

To characterize the effects of Mxc on estrogen responsive uterine genes,
five different experiments were conducted:

-   *Time course of LF and G6PD mRNA responses to Mxc*: Ovariectomized
    mice (WT and ERα-knockout) were injected with Mxc (at 15mg/kg BW).
    Tissues were collected at 2, 6, 12, 18, and 24 hours after
    treatment. LF and G6PD mRNA levels were quantified by competitive
    RT-PCR.

-   *Effects of different doses of Mxc on uterine LF mRNA
    concentrations:* WT and ERα-knockout mice were injected with Mxc
    (test doses were 3.75, 5.7, 7.5, 10.5, 15, 22, 30, 45, and 60 mg/kg
    BW; two consecutive identical injections 6 hours apart then tissue
    harvested 12 hours after final injection). LF mRNA levels were
    quantified by competitive RT-PCR.

-   *Effects of different doses of Mxc on uterine G6PD mRNA
    concentrations:* WT and ERα-knockout mice were injected with Mxc (9
    different test doses; two consecutive identical injections 6 hours
    apart with tissue harvested 12 hours after final injection). G6PD
    mRNA levels were quantified by competitive RT-PCR..

-   *Effects of estradiol-17β and ICI on Mxc-induced increases in LF
    mRNA concentrations:* In WT mice, nine treatment regimes were tested
    for effects on LF mRNA levels: oil control, Mxc (15 mg/kg BW),
    estradiol-17β (10μg/kg BW), estradiol-17β (10μg/kg BW) followed by
    Mxc, estradiol-17 (100μg/kg BW), estradiol-17 (100μg/kg BW) followed
    by Mxc, ICI[^1], ICI followed by Mxc, ICI followed by estradiol-17β
    (10μg/kg BW). In ERα-knockout mice, 8 treatment regimes were tested.
    The same treatment regimes as for WT except that ICI followed by
    estradiol-17β (10μg/kg BW) was excluded because neither
    estradiol-17βnor ICI simulate LF mRNA production and the combination
    of the two would not be expected to yield results different from
    either used alone. LF mRNA levels were quantified by competitive
    RT-PCR..

-   *Effects of estradiol-17β and ICI on Mxc-induced increases in G6PD
    mRNA concentrations:* The same treatment regimes used for LF were
    also used for G6PD analysis.

# Project Results and Implications  {#project-results-and-implications .LRI-Heading-2}

The first objective to characterize the effects of Mxc on estrogen
responsive uterine genes, was accomplished and the results were
published (Endocrinology 1999;140(8);3526-3533). The combined results
from the five experiments described above support a novel pathway for
Mxc signal transduction which is distinct but interacts with the ERα and
ERβ mediated pathways.

In the time course experiments a 2-fold increase in LF mRNA was seen 12
hours after treatment with Mxc, followed by a 7-fold increase (peak) at
18 hours and a decline to a 5-fold increase at 24 hours. No differences
between WT and ERα-knockout mice were observed for LF mRNA levels in
this time course experiment. A 2-fold increase in G6PD mRNA was seen 6
hours after treatment in WT mice and a 3.8-fold increase was seen 6
hours after treatment in ERα-knockout mice. In WT mice, a 3-fold
increase in G6PD mRNA was observed at 12 and 18 hours after treatment,
and a peak of 6-fold at 24 hours after treatment. In ERα-knockout mice,
a 6-fold increase in G6PD mRNA was observed at 12 and 18 hours after
treatment, and a peak of 9-fold at 24 hours after treatment. These
results indicate G6PD is an "early response" gene (within 6 hours) when
compared to LF (within 12 hours) and that Mxc acts on these two genes
through two different mechanisms, one more rapid than the other.

A biphasic dose-response curve was seen in both WT and ERα-knockout
mice, which suggests that more than one Mxc response mechanism is
active. The dose and time dependent response to Mxc supports its action
through receptor binding and saturation. The dose-response results
indicated that a dose of 15 mg/kg BW would be most effective for the
competitor experiments.

The results of the competition experiments are summarized in Figure 1A
and 1B below.

![](media/image1.png){width="3.25in" height="2.4166666666666665in"}
![](media/image2.png){width="3.123611111111111in"
height="2.313888888888889in"}

In ERα-knockout mice, the response to Mxc differs from the response to
estradiol-17β. In ERα-knockout mice, LF and G6PD mRNA levels are
stimulated by the addition of Mxc, but not by the addition of
estradiol-17β This indicates that Mxc action is not being mediated
solely through ERα. In wild type mice, Mxc and estradiol-17βstimulate
increases in LF and G6PD mRNA levels. This stimulation by estradiol-17β
can be inhibited by pretreatment with the antiestrogen ICI, but the
stimulation by Mxc is not inhibited by ICI. This finding supports a Mxc
signaling pathway that is independent of ERα and ERβ The combined
effects of Mxc and estradiol-17β on lactoferrin mRNA levels are not
additive. This indicates Mxc may act as a mixed agonist/antagonist to
estradiol-17β action by partial binding with ERα, as well as acting
through another pathway, or a Mxc-receptor complex could be an ERα
repressor. The combined effects of Mxc and estradiol-17β on G6PD mRNA
appear to be additive (within experimental error) at a 10μg/kg BW dose
of estradiol-17α, but not at 100μg/kg BW of estradiol-17β. The
variability in the10μg/kg BW of estradiol-17β followed Mxc treatment
group was greater than was seen for the other test groups. The reason
for this unusually high variability was not determined.

The different responses between estradiol-17β and Mxc in ERα-knockout
mice supports the idea that there is a Mxc signal transduction pathway
that is independent of ERα and ERβ activity. However, Mxc also interacts
with the ERα and ERβ signaling pathway, but the nature of that
interaction is not understood. The different timing of the responses of
LF and G6PD mRNA regulation due to exposure to Mxc supports the
likelihood that Mxc regulated LF and G6PD through different mechanisms.
The research results add to our growing knowledge of the complexity of
estrogen receptor signaling pathways and their interaction with
xenoestrogens.

The second objective, to identify the putative Mxc receptor, was not
completed under this grant, but additional research was "ongoing" at the
time the Final Report was issued. The low-stringency screening of cDNA
libraries had not yielded promising candidate genes. The human ERα, ERβ,
ERR1,and ERR3 receptors did not bind to Mxc in the cell lines studied.
Expression cloning was abandoned after in vivo experiments indicated
that Mxc is likely to have high non-specific binding, which would result
in a high rate of false positives.

# Investigators {#investigators .LRI-Heading-2}

Dennis B. Lubahn - University of Missouri - Columbia

Wade V. Welshons - University of Missouri - Columbia

# For More Information {#for-more-information .LRI-Heading-2}

**Laboratory web page:**

<http://www.biochem.missouri.edu/dlubahn.php>

<http://www.cvm.missouri.edu/vbms/index.html>

**NCER Project Abstract and Reports:**

<http://cfpub2.epa.gov/ncer_abstracts/index.cfm/fuseaction/display.abstractDetail/abstract/134/report/0>

[^1]: ICI-182780 (7
    alpha-\[9-(4,4,5,5,5-pentafluoropentylsulfinyl)nonyl\]estra-1,3,5,(1
    0)-triene-3,17 beta-diol)