Estrogens in hormone replacement therapy (HRT) decrease menopausal symptoms, but increase the risks of reproductive cancers. The beneficial effects of estrogen on peripheral tissues and the adverse proliferative effects on the uterus and mammary gland are mediated by ERalpha. Currently HRT is approved only for short-term use. Short-term HRT works for decreasing symptoms associated with menopause, however, long-term usage is needed to prevent subclinical diseases. Because estradiol-bound ERalpha is an agonist in all tissues there is a need for development of more tissue-selective estrogens that can be used for both short and long-term HRT. Chalcones display antiproliferative activity through ERbeta and may have benefits on menopause-induced hot flashes, however activity through ERalpha and their effects on both estradiol gene regulation and physiology are less known. The present study aimed at identifying a chalcone compound which could change the activity of ERalpha in the presence of estradiol as a coagonist, thereby modulating the response of ERalpha on gene regulation and increasing its tissue specificity. After screening a panel of five chalcone compounds for estrogenic activity in cells cotransfected with ERalpha and an ERE upstream of tk-luciferase, 2', 3', 4'-trihydroxychalcone was identified as a unique ERalpha coagonist. 2', 3', 4'-THC displayed no estrogenic activity on its own, but synergized the activation of the ERE in the presence of estradiol. Competitive binding assays with [3H]-estradiol demonstrated that 2', 3', 4'-THC binds to both ERalpha and ERbeta. Estradiol and SERM-induced genes, KRT-19 and NKG2E, were not regulated by 2', 3', 4'-THC alone. Both KRT-19 and NKG2E were synergized with the combination of 2', 3', 4'-THC and estradiol. Tamoxifen and raloxifene induced expression of NKG2E, but did not synergize the expression in the presence of estradiol. The data demonstrates that 2', 3', 4'-THC behaves as a novel coagonist and not a SERM on gene regulation. A unique gene expression profile was induced in U2OSalpha cells treated with a combination of estradiol and 2', 3', 4'-THC for 24 hours with doses that would allow binding of both ligands to ERalpha at the same time. Functional analysis utilizing the binding affinities of estradiol, 2', 3', 4'-THC and another ERalpha binding chalcone, 2, 2', 4'-THC, demonstrated that a heteroligand complex consisting of estradiol and 2', 3', 4'-THC in ERalpha is possible. Despite the synergistic activation of estradiol regulated genes in U2OSalpha cells, the combination of 2', 3', 4'-THC and estradiol did not induce proliferation of MCF-7 cells. In the same cells 2', 3', 4'-THC blocked estradiol-induced G1 to S phase cell cycle transition without blocking proliferative genes regulated by estradiol. In female ovariectomized mice on a soy-free chow diet treated for four weeks (n=5, per group), 2', 3', 4'-THC did not cause uterine proliferation and blocked estradiol-induced proliferation and gene expression. Although 2', 3', 4'-THC blocked estradiol gene expression in uterine tissue, it regulated and modulated estradiol-induced genes in adipose tissue. Because 2', 3', 4'-THC displays unique coagonist activity through ERalpha without causing proliferation, it may be useful for future HRT and expanding our knowledge of ERalpha regulation and ligand interaction.

Abstract

Conserved Molecular and Epigenetic Determinants of Aromatase Gene Induction by the Herbicide Atrazine in Human and Rat Cellular Models Relevant to Breast Cancer Risk

By

Theresa Ryan Stueve

Doctor of Philosophy in Molecular Toxicology

University of California, Berkeley

Professor Gary Firestone, Co-Chair

Professor Dale Leitman, Co-Chair

Fall 2011

The widely-applied herbicide atrazine (ATR) is a potent endocrine disruptor that elicits anti-androgenic and estrogenic effects, often at concentrations below the current drinking water standard (3 ppb), in wildlife and laboratory models of every vertebrate class studied to date. In oncogenicity studies conducted more than 15 years ago by its principle registrant, Ciby-Giecy (formerly Novartis Crop Protection, currently Syngenta), ATR increased plasma estradiol levels and mammary tumor incidence in ovary-intact but not ovariectomized (OVX) Sprague-Dawley (SD) rats. ATR inhibits phosphodiesterases and increases cAMP-signaling and steroidogenesis in several cell lines. Most relevant to rat and human mammary tumor risk, ATR induces expression of the enzyme aromatase (CYP19), which irreversibly converts androgens to estrogens, in several human and rat steroidogenic cell lines; however, ATR does not affect CYP19 in human estrogen-sensitive MCF-7 breast cancer cells. The significance of these findings to human health is debated and ATR is classified as 'not likely to be carcinogenic to humans' by the EPA due to 'lack of human and mechanistic data".

The mechanisms of rat mammary tumor induction by ATR have not been robustly interrogated for human relevance. Molecular determinants of ATR sensitivity in cell lines relevant to breast cancer (BC) risk, particularly fibroblast stromal cells, are poorly defined. In the present work, we assessed the effect of ATR on cyp19 promoter activity, mRNA levels, and trans-acting factors with consensus binding sites in the highly conserved cAMP-responsive gonadal region of cyp19, and identify conserved molecular and epigenetic determinants of CYP19 induction by ATR in human estrogen growth-sensitive BC cells (T-47D cell line), human adipose fibroblasts (SGBS cell line), and rat ovarian granulosa cells (SIRGC cell line).

ATR increased human gonadal cyp19 promoter activity in gene promoter-reporter studies by GATA, LRH-1, PKA- and PI3K-mediated mechanisms in human T-47D, SGBS, and significantly, rat SIRGC cells. ATR also increased endogenous Cyp19 mRNA levels in SGBS fibroblast and SIRGC rat granulosa cells, but only increased transcripts in T-47D cells pre-treated with the DNA-methylation inhibitor, 5-aza-dC.

Attempts to reconcile robust and conserved cAMP-responsive gonadal cyp19 promoter activation with varied mRNA-level effects in three alternately-responsive cell lines led us to the discovery that previously unknown epigenetic determinants are critical in conferring susceptibility to aberrant aromatase induction by ATR. We propose sensitivity to this widespread estrogenic herbicide varies inversely with Cyp19 gonadal promoter methylation, and directly with expression of GATA-3/4 and LRH-1/SF-1 transcription factors. Interrogating in other models the determinants of responsiveness we here define may enhance our understanding of how reported inter -strain, -species, and -laboratory differences in sensitivity can best inform risk assessments that account for human inter-individual susceptibility to gene-environment -related estrogen disorders.

We report a case of adult pyloric obstruction caused by the delayed presentation of a congenital gastric diverticulum. The derivation, classification and treatment of these abnormalities are discussed.