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

Abstract/nConserved Molecular and Epigenetic Determinants of Aromatase Gene Induction by the Herbicide Atrazine in Human and Rat Cellular Models Relevant to Breast Cancer Risk/nBy/nTheresa Ryan Stueve/nDoctor of Philosophy in Molecular Toxicology/nUniversity of California, Berkeley/nProfessor Gary Firestone, Co-Chair/nProfessor Dale Leitman, Co-Chair/nFall 2011/nThe 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"./nThe 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)./nATR 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./nAttempts 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.

Advisor: Leitman, Dale
Firestone, Gary L