##### Dioxin and Steroid Regulation in an Endometriosis Model

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

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-*p*-dioxin
(TCDD)---an unintentional by-product of several industrial chemical
processes and waste combustion, and one of the more potent and well
studied of a family of compounds referred to as dioxins---has been shown
by numerous laboratories to disrupt estrogen action in reproductive
tissues. TCDD also has been linked to the development in primates of
endometriosis, a persistent, steroid-mediated disease that develops in
10-15 percent of women in industrialized nations, most often from the
ectopic implantation of endometrial fragments that enter the peritoneum
by retrograde menstruation. This link between TCDD and endometriosis,
however, appears to be in conflict with TCDD\'s known anti-estrogenic
effects, and thus mechanistic basis for the association between TCDD
exposures and endometriosis in humans has been uncertain. Research under
this grant was aimed at supplementing the epidemiological information in
exposed human populations by studying the action of TCDD on specific
cellular characteristics linked to the pathophysiology of endometriosis.

Researchers hypothesized that, as a consequence of TCDD exposure,
progesterone-mediated regulation of matrix metalloproteinases (MMPs)---a
group of enzymes that help regulate the site and extent of connective
tissue remodeling throughout the body---may be disrupted. These
researchers have previously demonstrated, using a nude mouse model to
examine ectopic growth of human endometrium, that estrogen-associated
MMP expression supports development of experimental endometriosis, while
progesterone-mediated suppression of these enzymes inhibits the
development of ectopic lesions. Although TCDD clearly has an impact on
endometrial tissue function via interference or modulation of estrogen
action, the ability of this agent to affect progesterone action is less
understood. Thus, this grant sought to determine if and how TCDD can
interfere with progesterone-mediated MMP regulation via modulation of
local endometrial factors. Additionally, it sought to determine if this
interference plays a role in the establishment or progression of
TCDD-associated endometriosis.

The main objectives of this research were to:

-   Compare the expression patterns of several cell-specific MMPs in
    endometriotic lesions obtained from women with those seen in
    experimental endometriosis in the nude mouse model;

-   Identify the mechanisms by which TCDD exposure can disrupt
    progesterone-mediated regulation of endometrial MMPs; and

-   Investigate the molecular role of TCDD in altering cell-type
    specific response of endometrial MMP expression within the
    environment of the peritoneal cavity.

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

-   [Analysis of *In Vivo* MMP Expression Patterns]{.underline}: This
    study identified by *in situ* hybridization a highly variable
    expression pattern of MMP-3, MMP-7, and MMP-11 mRNA in endometriotic
    lesions obtained from different ectopic sites of disease growth.
    Although progesterone is normally a potent suppressor of
    cell-specific mRNAs for MMP-3, MMP-7, or MMP-11, expression of each
    gene, as well as protein secretion *in vitro*, was significantly
    increased in endometriosis tissues compared to the normal donor
    population. This finding is similar to observations in normal
    endometria that have been treated *in vivo* with TCDD.

-   [Cellular Mechanisms of TCDD Action]{.underline}: This set of
    experiments first explored the impact of TCDD on key elements of
    endometrial maturation during the menstrual cycle. Preliminary
    observations indicated that exposing human endometrial cell cultures
    to TCDD can prevent progesterone suppression of MMP-3 or MMP-7,
    which occurs *in vivo* during the progesterone-dominated secretory
    phase of the menstrual cycle. Because progesterone suppression of
    MMPs may be critical to prevention of experimental endometriosis,
    this study attempted to ascertain the specific cellular mechanism by
    which TCDD prevents progesterone suppression of MMPs. Progesterone
    induction of transforming growth factor (TGF-2) is known to be a
    critical step in the pathway by which progesterone mediates
    suppression of MMP-7 expression, and blocking of TGF-2 activity
    affects patterns of MMP-3 expression. During the first year of the
    grant, researchers were able to demonstrate that antibody treatments
    that block TGF-2 action greatly reduced the ability of progesterone
    treatments to prevent experimental endometriosis. Furthermore, they
    demonstrated that *in vitro* TCDD exposure prevented the increase in
    endometrial TGF-2 protein expression that normally follows exposure
    to progesterone. In subsequent experiments, researchers demonstrated
    that TCDD inhibition of TGF-2 occurs at the level of the gene
    expression because progesterone- associated increases in TGF-2 mRNA
    are also blocked by TCDD. Additionally, researchers found that
    disrupting TGF-2 expression dramatically increases endometrial MMP
    expression during the establishment of experimental endometriosis.
    These results indicate that the ability of TCDD to prevent
    progesterone induction of TGF-β2 may play a critical role in the
    toxin's ability to alter the normal pathway by which progesterone
    regulates MMP expression.

-   [Molecular Mechanisms of TCDD Action]{.underline}. The aryl
    hydrocarbon (AhR) and aryl hydrocarbon receptor nuclear translocator
    (Arnt) genes are known to modulate progesterone activity. To
    investigate the effects of TCDD on endometrial cells at the
    molecular level, experiments were conducted to test the effects of
    AhR and/or Arnt expression on the activity of a
    progesterone-responsive promoter. AhR was shown to be present in
    stromal cells maintained with progesterone, but it could not be
    determined if progesterone or decidualization plays a role in AhR
    regulation. In a second series of experiments, the effect of TCDD
    exposure on endometrial stromal cell expression of progesterone
    receptor isoforms A and B (PR-A, PR-B) was investigated. The
    experiments showed that TCDD disrupts the expression of the two
    isoforms, resulting in a decreased PR-B/PR-A ratio. The results are
    significant because an altered PR ratio (increased PR-A; decreased
    PR-B) would lead to diminished progesterone action, likely affecting
    the expression of additional TGF- signaling molecules. For example,
    the researchers found decreased PR-B/PR-A ratios in the endometria
    of women with endometriosis. Additionally, immunohistochemical
    analysis of endometria from women with endometriosis revealed a near
    absence of differentiation-related expression of TGF-β2, again
    reflecting the finding following *in vitro* TCDD exposure.

In summary, this grant has contributed substantially to the
understanding of whether and how TCDD affects progesterone-mediated MMP
regulation via modulation of local endometrial factors, as well as
clarifying the role of TCDD in the establishment and progression of
endometriosis. This grant also demonstrated the benefits of an
endometriosis model involving the ectopic implantation of human
endometrial tissue in nude mice.

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

Kevin G. Osteen -- Women's Reproductive Health Research Center,
Vanderbilt University School of Medicine, Nashville, TN

Kaylon L. Bruner-Tran - Women's Reproductive Health Research Center,
Vanderbilt University School of Medicine, Nashville, TN

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

Women's Reproductive Health Research Center, Vanderbilt University
School of Medicine

<http://www.mc.vanderbilt.edu/root/vumc.php?site=wrhrc>

NCER Project Abstract and Reports

<http://cfpub2.epa.gov/ncer_abstracts/index.cfm/fuseaction/display.abstractDetail/abstract/171/report/0>

<http://cfpub.epa.gov/ncer_abstracts/index.cfm/fuseaction/display.abstractDetail/abstract/171/report/1999>

<http://cfpub.epa.gov/ncer_abstracts/index.cfm/fuseaction/display.abstractDetail/abstract/171/report/F>