n-6 High Fat Diet Induces Gut Microbiome Dysbiosis and Colonic Inflammation.

Background: Concerns are emerging that a high-fat diet rich in n-6 PUFA (n-6HFD) may alter gut microbiome and increase the risk of intestinal disorders. Research is needed to model the relationships between consumption of an n-6HFD starting at weaning and development of gut dysbiosis and colonic inflammation in adulthood. We used a C57BL/6J mouse model to compare the effects of exposure to a typical American Western diet (WD) providing 58.4%, 27.8%, and 13.7% energy (%E) from carbohydrates, fat, and protein, respectively, with those of an isocaloric and isoproteic soybean oil-rich n-6HFD providing 50%E and 35.9%E from total fat and carbohydrates, respectively on gut inflammation and microbiome profile. Methods: At weaning, male offspring were assigned to either the WD or n-6HFD through 10-16 weeks of age. The WD included fat exclusively from palm oil whereas the n-6HFD contained fat exclusively from soybean oil. We recorded changes in body weight, cyclooxygenase-2 (COX-2) expression, colon histopathology, and gut microbiome profile. Results: Compared to the WD, the n-6HFD increased plasma levels of n-6 fatty acids; colonic expression of COX-2; and the number of colonic inflammatory and hyperplastic lesions. At 16 weeks of age, the n-6HFD caused a marked reduction in the gut presence of Firmicutes, Clostridia, and Lachnospiraceae, and induced growth of Bacteroidetes and Deferribacteraceae. At the species level, the n-6HFD sustains the gut growth of proinflammatory Mucispirillum schaedleri and Lactobacillus murinus. Conclusions: An n-6HFD consumed from weaning to adulthood induces a shift in gut bacterial profile associated with colonic inflammation.

A Villin-Driven Fxr Transgene Modulates Enterohepatic Bile Acid Homeostasis and Response to an n-6-Enriched High-Fat Diet.

A diet high in n-6 polyunsaturated fatty acids (PUFAs) may contribute to inflammation and tissue damage associated with obesity and pathologies of the colon and liver. One contributing factor may be dysregulation by n-6 fatty acids of enterohepatic bile acid (BA) metabolism. The farnesoid X receptor (FXR) is a nuclear receptor that regulates BA homeostasis in the liver and intestine. This study aims to compare the effects on FXR regulation and BA metabolism of a palm oil-based diet providing 28% energy (28%E) from fat and low n-6 linoleic acid (LA, 2.5%E) (CNTL) with those of a soybean oil-based diet providing 50%E from fat and high (28%E) in LA (n-6HFD). Wild-type (WT) littermates and a transgenic mouse line overexpressing the Fxrα1 isoform under the control of the intestine-specific Villin promoter (Fxrα1TG) were fed the CNTL or n-6HFD starting at weaning through 16 weeks of age. Compared to the CNTL diet, the n-6HFD supports higher weight gain in both WT and FxrαTG littermates; increases the expression of Fxrα1/2, and peroxisome proliferator-activated receptor-γ1 (Pparγ1) in the small intestine, Fxrα1/2 in the colon, and cytochrome P4507A1 (Cyp7a1) and small heterodimer protein (Shp) in the liver; and augments the levels of total BA in the liver, and primary chenodeoxycholic (CDCA), cholic (CA), and ß-muricholic (ßMCA) acid in the cecum. Intestinal overexpression of the Fxra1TG augments expression of Shp and ileal bile acid-binding protein (Ibabp) in the small intestine and Ibabp in the proximal colon. Conversely, it antagonizes n-6HFD-dependent accumulation of intestinal and hepatic CDCA and CA; hepatic levels of Cyp7a1; and expression of Pparγ in the small intestine. We conclude that intestinal Fxrα1 overexpression represses hepatic de novo BA synthesis and protects against n-6HFD-induced accumulation of human-specific primary bile acids in the cecum.

Aryl Hydrocarbon Receptor Diet and Breast Cancer Risk.

Breast cancer is the most common type of cancer and leading cause of cancer mortality among women worldwide. However, the majority of breast malignancies are of sporadic etiology. Therefore, identifying risk-mitigating factors may significantly decrease the burden of breast cancer. Diet can have both a predisposing and protective role in breast tumorigenesis. However, establishing efficacy of dietary constituents for cancer prevention has been limited by suboptimal dietary assessment. There is a need to acquire new experimental evidence that can be used to discriminate beneficial from harmful dietary constituents. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is recognized as the mediator of halogenated and polycyclic aromatic hydrocarbon toxicities. Importantly, evidence points to a breast tumor-promoting role for the AhR. Preclinical and clinical studies suggest that the AhR is overexpressed in advanced and triple negative breast cancers. Several dietary constituents, namely flavonoid compounds, have demonstrated inhibitory effects on AhR activation. Given this background, in this paper we elaborate on the working hypothesis that a diet rich in AhR food agonists favors breast tumor development, whereas a diet rich in AhR food antagonists is protective. As an initial approach to developing an AhR diet hypothesis, we conducted a review of published studies reporting on the association between intake of AhR inhibitory foods and risk of breast cancer. To assist the reader with interpretation of the concepts leading to the AhR diet hypothesis, we have preceded this review with an overview of AhR biology and its role in breast cancer development.

Inactivation of Adenomatous Polyposis Coli Reduces Bile Acid/Farnesoid X Receptor Expression through Fxr gene CpG Methylation in Mouse Colon Tumors and Human Colon Cancer Cells.

BACKGROUND: The farnesoid X receptor (FXR) regulates bile acid (BA) metabolism and possesses tumor suppressor functions. FXR expression is reduced in colorectal tumors of subjects carrying inactivated adenomatous polyposis coli (APC). Identifying the mechanisms responsible for this reduction may offer new molecular targets for colon cancer prevention. OBJECTIVE: We investigated how APC inactivation influences the regulation of FXR expression in colonic mucosal cells. We hypothesized that APC inactivation would epigenetically repress nuclear receptor subfamily 1, group H, member 4 (FXR gene name) expression through increased CpG methylation. METHODS: Normal proximal colonic mucosa and normal-appearing adjacent colonic mucosa and colon tumors were collected from wild-type C57BL/6J and Apc-deficient (Apc(Min) (/+)) male mice, respectively. The expression of Fxr, ileal bile acid-binding protein (Ibabp), small heterodimer partner (Shp), and cyclooxygenase-2 (Cox-2) were determined by real-time polymerase chain reaction. In both normal and adjacent colonic mucosa and colon tumors, we measured CpG methylation of Fxr in bisulfonated genomic DNA. In vitro, we measured the impact of APC inactivation and deoxycholic acid (DCA) treatment on FXR expression in human colon cancer HCT-116 cells transfected with silencing RNA for APC and HT-29 cells carrying inactivated APC. RESULTS: In Apc(Min) (/+) mice, constitutive CpG methylation of the Fxrα3/4 promoter was linked to reduced (60-90%) baseline Fxr, Ibabp, and Shp and increased Cox-2 expression in apparently normal adjacent mucosa and colon tumors. Apc knockdown in HCT-116 cells increased cellular myelocytomatosis (c-MYC) and lowered (∼50%) FXR expression, which was further reduced (∼80%) by DCA. In human HCT-116 but not HT-29 colon cancer cells, DCA induced FXR expression and lowered CpG methylation of FXR. CONCLUSIONS: We conclude that the loss of APC function favors the silencing of FXR expression through CpG hypermethylation in mouse colonic mucosa and human colon cells, leading to reduced expression of downstream targets (SHP, IBABP) involved in BA homeostasis while increasing the expression of factors (COX-2, c-MYC) that contribute to inflammation and colon cancer.

Gestational exposure to the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin induces BRCA-1 promoter hypermethylation and reduces BRCA-1 expression in mammary tissue of rat offspring: preventive effects of resveratrol.

Studies with murine models suggest that maternal exposure to aromatic hydrocarbon receptor (AhR) agonists may impair mammary gland differentiation and increase the susceptibility to mammary carcinogenesis in offspring. However, the molecular mechanisms responsible for these perturbations remain largely unknown. Previously, we reported that the AhR agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced CpG methylation of the breast cancer-1 (BRCA-1) gene and reduced BRCA-1 expression in breast cancer cell lines. Based on the information both the human and rat BRCA-1 genes harbor xenobiotic responsive elements (XRE = 5'-GCGTG-3'), which are binding targets for the AhR, we extended our studies to the analysis of offspring of pregnant Sprague-Dawley rats treated during gestation with TCDD alone or in combination with the dietary AhR antagonist resveratrol (Res). We report that the in utero exposure to TCDD increased the number of terminal end buds (TEB) and reduced BRCA-1 expression in mammary tissue of offspring. The treatment with TCDD induced occupancy of the BRCA-1 promoter by DNA methyltransferase-1 (DNMT-1), CpG methylation of the BRCA-1 promoter, and expression of cyclin D1 and cyclin-dependent kinase-4 (CDK4). These changes were partially overridden by pre-exposure to Res, which stimulated the expression of the AhR repressor (AhRR) and its recruitment to the BRCA-1 gene. These findings point to maternal exposure to AhR agonists as a risk factor for breast cancer in offspring through epigenetic inhibition of BRCA-1 expression, whereas dietary antagonists of the AhR may exert protective effects.

Constitutive expression of AhR and BRCA-1 promoter CpG hypermethylation as biomarkers of ERα-negative breast tumorigenesis.

BACKGROUND: Only 5-10% of breast cancer cases is linked to germline mutations in the BRCA-1 gene and occurs early in life. Conversely, sporadic breast tumors, which represent 90-95% of breast malignancies, have lower BRCA-1 expression, but not mutated BRCA-1 gene, and tend to occur later in life in combination with other genetic alterations and/or environmental exposures. The latter may include environmental and dietary factors that activate the aromatic hydrocarbon receptor (AhR). Therefore, understanding if changes in expression and/or activation of the AhR are associated with somatic inactivation of the BRCA-1 gene may provide clues for breast cancer therapy. METHODS: We evaluated Brca-1 CpG promoter methylation and expression in mammary tumors induced in Sprague-Dawley rats with the AhR agonist and mammary carcinogen 7,12-dimethyl-benzo(a)anthracene (DMBA). Also, we tested in human estrogen receptor (ER)α-negative sporadic UACC-3199 and ERα-positive MCF-7 breast cancer cells carrying respectively, hyper- and hypomethylated BRCA-1 gene, if the treatment with the AhR antagonist α-naphthoflavone (αNF) modulated BRCA-1 and ERα expression. Finally, we examined the association between expression of AhR and BRCA-1 promoter CpG methylation in human triple-negative (TNBC), luminal-A (LUM-A), LUM-B, and epidermal growth factor receptor-2 (HER-2)-positive breast tumor samples. RESULTS: Mammary tumors induced with DMBA had reduced BRCA-1 and ERα expression; higher Brca-1 promoter CpG methylation; increased expression of Ahr and its downstream target Cyp1b1; and higher proliferation markers Ccnd1 (cyclin D1) and Cdk4. In human UACC-3199 cells, low BRCA-1 was paralleled by constitutive high AhR expression; the treatment with αNF rescued BRCA-1 and ERα, while enhancing preferential expression of CYP1A1 compared to CYP1B1. Conversely, in MCF-7 cells, αNF antagonized estradiol-dependent activation of BRCA-1 without effects on expression of ERα. TNBC exhibited increased basal AhR and BRCA-1 promoter CpG methylation compared to LUM-A, LUM-B, and HER-2-positive breast tumors. CONCLUSIONS: Constitutive AhR expression coupled to BRCA-1 promoter CpG hypermethylation may be predictive markers of ERα-negative breast tumor development. Regimens based on selected AhR modulators (SAhRMs) may be useful for therapy against ERα-negative tumors, and possibly, TNBC with increased AhR and hypermethylated BRCA-1 gene.

Resveratrol prevents epigenetic silencing of BRCA-1 by the aromatic hydrocarbon receptor in human breast cancer cells.

The BRCA-1 protein is a tumor suppressor involved in repair of DNA damage. Epigenetic mechanisms contribute to its reduced expression in sporadic breast tumors. Through diet, humans are exposed to a complex mixture of xenobiotics and natural ligands of the aromatic hydrocarbon receptor (AhR), which contributes to the etiology of various types of cancers. The AhR binds xenobiotics, endogenous ligands, and many natural dietary bioactive compounds, including the phytoalexin resveratrol (Res). In estrogen receptor- alpha (ER alpha )-positive and BRCA-1 wild-type MCF-7 breast cancer cells, we investigated the influence of AhR activation with the agonist 2,3,7,8 tetrachlorobenzo(p)dioxin (TCDD) on epigenetic regulation of the BRCA-1 gene and the preventative effects of Res. We report that activation and recruitment of the AhR to the BRCA-1 promoter hampers 17 beta -estradiol (E2)-dependent stimulation of BRCA-1 transcription and protein levels. These inhibitory effects are paralleled by reduced occupancy of ER alpha , acetylated histone (AcH)-4, and AcH3K9. Conversely, the treatment with TCDD increases the association of mono-methylated-H3K9, DNA-methyltransferase-1 (DNMT1), and methyl-binding domain protein-2 with the BRCA-1 promoter and stimulates the accumulation of DNA strand breaks. The AhR-dependent repression of BRCA-1 expression is reversed by small interference for the AhR and DNMT1 or pretreatment with Res, which reduces TCDD-induced DNA strand breaks. These results support the hypothesis that epigenetic silencing of the BRCA-1 gene by the AhR is preventable with Res and provide the molecular basis for the development of dietary strategies based on natural AhR antagonists.

Gene expression profiling in the fetal cardiac tissue after folate and low-dose trichloroethylene exposure.

BACKGROUND: Previous studies show gene expression alterations in rat embryo hearts and cell lines that correspond to the cardio-teratogenic effects of trichloroethylene (TCE) in animal models. One potential mechanism of TCE teratogenicity may be through altered regulation of calcium homeostatic genes with a corresponding inhibition of cardiac function. It has been suggested that TCE may interfere with the folic acid/methylation pathway in liver and kidney and alter gene regulation by epigenetic mechanisms. According to this hypothesis, folate supplementation in the maternal diet should counteract TCE effects on gene expression in the embryonic heart. APPROACH: To identify transcriptional targets altered in the embryonic heart after exposure to TCE, and possible protective effects of folate, we used DNA microarray technology to profile gene expression in embryonic mouse hearts with maternal TCE exposure and dietary changes in maternal folate. RESULTS: Exposure to low doses of TCE (10 ppb) caused extensive alterations in transcripts encoding proteins involved in transport, ion channel, transcription, differentiation, cytoskeleton, cell cycle, and apoptosis. Exogenous folate did not offset the effects of TCE exposure on normal gene expression, and both high and low levels of folate produced additional significant changes in gene expression. CONCLUSIONS: A mechanism by which TCE induces a folate deficiency does not explain altered gene expression patterns in the embryonic mouse heart. The data further suggest that use of folate supplementation, in the presence of this toxin, may be detrimental and not protective of the developing embryo.

HNF4a transcription is a target of trichloroethylene toxicity in the embryonic mouse heart.

In exploration of congenital heart defects produced by TCE, Hepatocyte Nuclear Factor 4 alpha (HNF4a) transcriptional activity was identified as a central component. TCE exposure altered gene transcription in the chick heart in a non-monotonic pattern where only low dose exposure inhibited transcription by HNF4a. As the chick embryo is non-placental, we examine here HNF4a as a target of TCE in developing mouse embryos. Benfluorex and Bi6015, published agonist and antagonist, respectively, of HNF4a were compared to low dose TCE exposure. Pregnant mice were exposed to 10 ppb (76 nM) TCE, 5 µM Benfluorex, 5 µM Bi6015, or a combination of Bi6015 and TCE in drinking water. Litters (E12) were collected during a sensitive window in heart development. Embryonic hearts were collected, pooled for extraction of RNA and marker expression was examined by quantitative PCR. Multiple markers, previously identified as sensitive to TCE exposure in chicks or as published targets of HNF4a transcription were significantly affected by Benfluorex, Bi6015 and TCE. Activity of TCE and both HNF4a-specific reagents on transcription argues that HNF4a is a component of TCE cardiotoxicity and likely a proximal target of low dose exposure during development. The effectiveness of these reagents after delivery in maternal drinking water suggests that neither maternal metabolism, nor placental transport is protective of exposure.

Dietary fat and obesity as modulators of breast cancer risk: Focus on DNA methylation.

Breast cancer (BC) is the most common cancer and second leading cause of cancer mortality in women worldwide. Validated biomarkers enhance efforts for early detection and treatment, which reduce the risk of mortality. Epigenetic signatures have been suggested as good biomarkers for early detection, prognosis and targeted therapy of BC. Here, we highlight studies documenting the modifying effects of dietary fatty acids and obesity on BC biomarkers associated with DNA methylation. We focus our analysis on changes elicited in writers of DNA methylation (i.e., DNA methyltransferases), global DNA methylation and gene-specific DNA methylation. To provide context, we precede this discussion with a review of the available evidence for an association between BC incidence and both dietary fat consumption and obesity. We also include a review of well-vetted BC biomarkers related to cytosine-guanine dinucleotides methylation and how they influence BC risk, prognosis, tumour characteristics and response to treatment. LINKED ARTICLES: This article is part of a themed section on The Pharmacology of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.6/issuetoc.

Epigenetic Regulation and Dietary Control of Triple Negative Breast Cancer.

Triple negative breast cancer (TNBC) represents a highly heterogeneous group of breast cancers, lacking expression of the estrogen (ER) and progesterone (PR) receptors, and human epidermal growth factor receptor 2 (HER2). TNBC are characterized by a high level of mutation and metastasis, poor clinical outcomes and overall survival. Here, we review the epigenetic mechanisms of regulation involved in cell pathways disrupted in TNBC, with particular emphasis on dietary food components that may be exploited for the development of effective strategies for management of TNBC.

Bisphenols and Risk of Breast Cancer: A Narrative Review of the Impact of Diet and Bioactive Food Components.

Data from preclinical studies suggest a link between increased risk of breast cancer and exposure to bisphenols at doses below what the United States Food and Drug Administration (FDA) considers as safe for consumption. Bisphenols exert estrogenic effects and are found in canned and plastic wrapped foods, food packaging, and plasticware. Mechanistically, bisphenols bind to the estrogen receptor (ER) and activate the expression of genes associated with cell proliferation and breast cancer. In this paper, we present a narrative literature review addressing bisphenol A and chemical analogs including bisphenol AF, bisphenol F, and bisphenol S selected as prototype xenoestrogens; then, we discuss biological mechanisms of action of these bisphenols in breast cells and potential impact of exposure at different stages of development (i.e., perinatal, peripubertal, and adult). Finally, we summarize studies detailing interactions, both preventative and promoting, of bisphenols with food components on breast cancer risk. We conclude the review with a discussion of current controversies in interpretation of the above research and future areas for investigation, including the impact of bisphenols and food components on breast tumor risk.

Do Olive and Fish Oils of the Mediterranean Diet Have a Role in Triple Negative Breast Cancer Prevention and Therapy? An Exploration of Evidence in Cells and Animal Models.

Breast cancer is the most common malignancy and cause of cancer-related mortality among women worldwide. Triple negative breast cancers (TNBC) are the most aggressive and lethal of the breast cancer molecular subtypes, due in part to a poor understanding of TNBC etiology and lack of targeted therapeutics. Despite advances in the clinical management of TNBC, optimal treatment regimens remain elusive. Thus, identifying interventional approaches that suppress the initiation and progression of TNBC, while minimizing side effects, would be of great interest. Studies have documented an inverse relationship between the incidence of hormone receptor negative breast cancer and adherence to a Mediterranean Diet, particularly higher consumption of fish and olive oil. Here, we performed a review of studies over the last 5 years investigating the effects of fish oil, olive oil and their components in model systems of TNBC. We included studies that focused on the fish oil ω-3 essential fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in addition to olive oil polyphenolic compounds and oleic acid. Both beneficial and deleterious effects on TNBC model systems are reviewed and we highlight how multiple components of these Mediterranean Diet oils target signaling pathways known to be aberrant in TNBC including PI3K/Akt/mTOR, NF-κB/COX2 and Wnt/ß-catenin.

Targeting of aryl hydrocarbon receptor-mediated activation of cyclooxygenase-2 expression by the indole-3-carbinol metabolite 3,3'-diindolylmethane in breast cancer cells.

Ligands of the aryl hydrocarbon receptor (AhR) include the environmental xenobiotic 2,3,7,8 tetrachlorodibenzo(p)dioxin (TCDD), polycyclic aryl hydrocarbons, and the dietary compounds 3, 3'-diindolylmethane (DIM), a condensation product of indol-3-carbinol found in Brassica vegetables, and the phytoalexin resveratrol (RES). The AhR and its cofactors regulate the expression of target genes at pentameric (GCGTG) xenobiotic responsive elements (XRE). Because the activation of cyclooxygenase-2 (COX-2) expression by AhR ligands may contribute to inflammation and tumorigenesis, we investigated the epigenetic regulation of the COX-2 gene by TCDD and the reversal effects of DIM in MCF-7 breast cancer cells. Results of DNA binding and chromatin immunoprecipitation (ChIP) studies documented that the treatment with TCDD induced the association of the AhR to XRE harbored in the COX-2 promoter and control CYP1A1 promoter oligonucleotides. The TCDD-induced binding of the AhR was reduced by small-interfering RNA for the AhR or the cotreatment with synthetic (3-methoxy-4-naphthoflavone) or dietary AhR antagonists (DIM, RES). In time course ChIP studies, TCDD induced the rapid (15 min) occupancy by the AhR, the histone acetyl transferase p300, and acetylated histone H4 (AcH4) at the COX-2 promoter. Conversely, the cotreatment of MCF-7 cells with DIM (10 micromol/L) abrogated the TCDD-induced recruitment of the AhR and AcH4 to the COX-2 promoter and the induction of COX-2 mRNA and protein levels. Taken together, these data suggest that naturally occurring modulators of the AhR such as DIM may be effective agents for dietary strategies against epigenetic activation of COX-2 expression by AhR agonists.

n-6 Linoleic Acid Induces Epigenetics Alterations Associated with Colonic Inflammation and Cancer.

The farnesoid-X-receptor (FXR) protects against inflammation and cancer of the colon through maintenance of intestinal bile acid (BA) homeostasis. Conversely, higher levels of BA and cyclooxygenase-2 (COX-2) are risk factors for inflammation and cancer of the colon. In the United States, n-6 linoleic acid (LA) is the most commonly used dietary vegetable fat. Metabolism of n-6 fatty acids has been linked to a higher risk of intestinal cancer. The objectives of this study were to investigate in colonic mucosa the effects of a high-fat diet rich in LA (n-6HFD) on CpG methylation of Fxr and prostaglandin-endoperoxide synthase-2 (Ptsg-2) genes, and the impact on the expression of tumor suppressor adenomatous polyposis Coli (Apc) and proliferative cyclin D1 (Ccnd1) genes. Weaned C57BL/6J male mice were fed for 6 weeks either an n-6HFD containing 44% energy (44%E) from 22% safflower oil (SO, 76% LA by weight) or a 13% energy (13%E) control diet (Control) from SO (5% by weight). Mice fed the n-6HFD had reduced (60%) Fxr promoter CpG methylation and increased (~50%) Fxr mRNA. The expression of FXR-target ileal bile acid-binding protein (Ibabp), small heterodimer protein (Shp), and anti-inflammatory peroxisome proliferator-activated-γ1 genes was increased. The n-6HFD reduced Ptgs-2 CpG methylation, increased the expression of Cox-2, and increased Apc CpG methylation in colonic mucosa. Accordingly, reduced expression of Apc was coupled to accumulation of c-JUN and Ccnd1, respectively cofactor and gene targets for the ß-catenin/Wnt signaling pathway. Finally, the n-6HFD reduced the expression of histone deacetylase-1 while favoring the accumulation of acetylated histone 3. We conclude that an n-6HFD epigenetically modifies Fxr, leading to the activation of downstream factors that participate in BA homeostasis. However, epigenetic activation of Ptsg-2 coupled with silencing of Apc and accumulation of C-JUN and Ccnd1 may increase the risk of inflammation and cancer of the colon.

Epigenetic Activation of BRCA1 by Genistein In Vivo and Triple Negative Breast Cancer Cells Linked to Antagonism toward Aryl Hydrocarbon Receptor.

Triple negative breast cancers (TNBC) are the most aggressive and lethal breast cancers (BC). The aryl hydrocarbon receptor (AHR) is often overexpressed in TNBC, and its activation results in the epigenetic silencing of BRCA1, which is a necessary factor for the transcriptional activation of estrogen receptor (ER)α. The dietary isoflavone genistein (GEN) modulates BRCA1 CpG methylation in BC cells. The purpose of this study was to investigate the effect of GEN on BRCA1 epigenetic regulation and AHR activity in vivo and TNBC cells. Mice were administered a control or GEN-enriched (4 and 10 ppm) diet from gestation through post-natal day 50. Mammary tissue was analyzed for changes in BRCA1 regulation and AhR activity. TNBC cells with constitutively hypermethylated BRCA1 (HCC38) and MCF7 cells were used. Protein levels and mRNA expression were measured by Western blot and real-time PCR, respectively. BRCA1 promoter occupancy and CpG methylation were analyzed by chromatin immunoprecipitation and methylation-specific PCR, respectively. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. GEN administered in the diet dose-dependently decreased basal Brca1 methylation and AHR activity in the mammary gland of adult mice. HCC38 cells were found to overexpress constitutively active AHR in parallel with BRCA1 hypermethylation. The treatment of HCC38 cells with GEN upregulated BRCA1 protein levels, which was attributable to decreased CpG methylation and AHR binding at BRCA1 exon 1a. In MCF7 cells, GEN prevented the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-dependent localization of AHR at the BRCA1 gene. These effects were consistent with those elicited by control AHR antagonists galangin (GAL), CH-223191, and α-naphthoflavone. The pre-treatment with GEN sensitized HCC38 cells to the antiproliferative effects of 4-hydroxytamoxifen. We conclude that the dietary compound GEN may be effective for the prevention and reversal of AHR-dependent BRCA1 hypermethylation, and the restoration of ERα-mediated response, thus imparting the sensitivity of TNBC to antiestrogen therapy.

Arsenic­induced BRCA1 CpG promoter methylation is associated with the downregulation of ERα and resistance to tamoxifen in MCF7 breast cancer cells and mouse mammary tumor xenografts.

A significant percentage (~30%) of estrogen receptor­α (ERα)­positive tumors become refractory to endocrine therapies; however, the mechanisms responsible for this resistance remain largely unknown. Chronic exposure to arsenic through foods and contaminated water has been linked to an increased incidence of several tumors and long­term health complications. Preclinical and population studies have indicated that arsenic exposure may interfere with endocrine regulation and increase the risk of breast tumorigenesis. In this study, we examined the effects of sodium arsenite (NaAsIII) exposure in ERα­positive breast cancer cells in vitro and in mammary tumor xenografts. The results revealed that acute (within 4 days) and long­term (10 days to 7 weeks) in vitro exposure to environmentally relevant doses reduced breast cancer 1 (BRCA1) and ERα expression associated with the gain of cyclin D1 (CCND1) and folate receptor 1 (FOLR1), and the loss of methylenetetrahydrofolate reductase (MTHFR) expression. Furthermore, long­term exposure to NaAsIII induced the proliferation and compromised the response of MCF7 cells to tamoxifen (TAM). The in vitro exposure to NaAsIII induced BRCA1 CpG methylation associated with the increased recruitment of DNA methyltransferase 1 (DNMT1) and the loss of RNA polymerase II (PolII) at the BRCA1 gene. Xenografts of NaAsIII­preconditioned MCF7 cells (MCF7NaAsIII) into the mammary fat pads of nude mice produced a larger tumor volume compared to tumors from control MCF7 cells and were more refractory to TAM in association with the reduced expression of BRCA1 and ERα, CpG hypermethylation of estrogen receptor 1 (ESR1) and BRCA1, and the increased expression of FOLR1. These cumulative data support the hypothesis that exposure to AsIII may contribute to reducing the efficacy of endocrine therapy against ERα­positive breast tumors by hampering the expression of ERα and BRCA1 via CpG methylation, respectively of ESR1 and BRCA1.

Trichloroethylene and trichloroacetic acid regulate calcium signaling pathways in murine embryonal carcinoma cells p19.

Trichloroethylene (TCE) and its metabolite trichloroacetic acid (TCA) are ubiquitous environmental contaminants which have been regarded as risk factors for congenital heart malformations. An increasing body of evidence from in vivo and in vitro studies supports the notion that exposure to TCE and TCA may interfere with normal embryonic heart development. The expression of several genes coding for factors implicated in the regulation of cardiac development has been shown to be modified by TCE or TCA, but the molecular mechanisms that mediate these effects are still obscure. In this study, we investigated the global changes in gene expression caused by exposure of P19 embryonal carcinoma cells to TCE and TCA, and whether or not TCE and/or TCA influence the expression levels of genes encoding for proteins that regulate calcium fluxes in cardiac cells. We report that TCE and TCA disrupt the expression of genes involved in processes important during embryonic development suggesting that exposure to environmentally significant concentrations of TCE may have deleterious effects on specific stages of cardiac differentiation.

The ligand status of the aromatic hydrocarbon receptor modulates transcriptional activation of BRCA-1 promoter by estrogen.

In sporadic breast cancers, BRCA-1 expression is down-regulated in the absence of mutations in the BRCA-1 gene. This suggests that disruption of BRCA-1 expression may contribute to the onset of mammary tumors. Environmental contaminants found in industrial pollution, tobacco smoke, and cooked foods include benzo(a)pyrene [B(a)P] and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which have been shown to act as endocrine disruptors and tumor promoters. In previous studies, we documented that estrogen (E2) induced BRCA-1 transcription through the recruitment of an activator protein-1/estrogen receptor-alpha (ER alpha) complex to the proximal BRCA-1 promoter. Here, we report that activation of BRCA-1 transcription by E2 requires occupancy of the BRCA-1 promoter by the unliganded aromatic hydrocarbon receptor (AhR). The stimulatory effects of E2 on BRCA-1 transcription are counteracted by (a) cotreatment with the AhR antagonist 3'-methoxy-4'-nitroflavone; (b) transient expression in ER alpha-negative HeLa cells of ER alpha lacking the protein-binding domain for the AhR; and (c) mutation of two consensus xenobiotic-responsive elements (XRE, 5'-GCGTG-3') located upstream of the ER alpha-binding region. These results suggest that the physical interaction between the unliganded AhR and the liganded ER alpha plays a positive role in E2-dependent activation of BRCA-1 transcription. Conversely, we show that the AhR ligands B(a)P and TCDD abrogate E2-induced BRCA-1 promoter activity. The repressive effects of TCDD are paralleled by increased recruitment of the liganded AhR and HDAC1, reduced occupancy by p300, SRC-1, and diminished acetylation of H4 at the BRCA-1 promoter region flanking the XREs. We propose that the ligand status of the AhR modulates activation of the BRCA-1 promoter by estrogen.

Trichloroethylene perturbs HNF4a expression and activity in the developing chick heart.

Exposure to trichloroethylene (TCE) is linked to formation of congenital heart defects in humans and animals. Prior interactome analysis identified the transcription factor, Hepatocyte Nuclear Factor 4 alpha (HNF4a), as a potential target of TCE exposure. As a role for HNF4a is unknown in the heart, we examined developing avian hearts for HNF4a expression and for sensitivity to TCE and the HNF4a agonist, Benfluorex. In vitro analysis using a HNF4a reporter construct showed both TCE and HFN4a to be antagonists of HNF4a-mediated transcription at the concentrations tested. HNF4a mRNA is expressed transiently in the embryonic heart during valve formation and cardiac development. Embryos were examined for altered gene expression in the presence of TCE or Benfluorex. TCE altered expression of selected mRNAs including HNF4a, TRAF6 and CYP2C45. There was a transition between inhibition and induction of marker gene expression in embryos as TCE concentration increased. Benfluorex was largely inhibitory to selected markers. Echocardiography of exposed embryos showed reduced cardiac function with both TCE and Benfluorex. Cardiac contraction was reduced by 29% and 23%, respectively at 10 ppb. The effects of TCE and Benfluorex on autocrine regulation of HNF4a, selected markers and cardiac function argue for a functional interaction of TCE and HNF4a. Further, the dose-sensitive shift between inhibition and induction of marker expression may explain the nonmonotonic-like dose response observed with TCE exposure in the heart.