Loss of aryl hydrocarbon receptor potentiates FoxM1 signaling to enhance self-renewal of colonic stem and progenitor cells.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that senses xenobiotics, diet, and gut microbial-derived metabolites, is increasingly recognized as a key regulator of intestinal biology. However, its effects on the function of colonic stem and progenitor cells remain largely unexplored. Here, we observed that inducible deletion of AhR in Lgr5+ stem cells increases the percentage of colonic stem cells and enhances organoid initiating capacity and growth of sorted stem and progenitor cells, while AhR activation has the opposite effect. Moreover, intestinal-specific AhR knockout increases basal stem cell and crypt injury-induced cell proliferation and promotes colon tumorigenesis in a preclinical colitis-associated tumor model by upregulating FoxM1 signaling. Mechanistically, AhR transcriptionally suppresses FoxM1 expression. Activation of AhR in human organoids recapitulates phenotypes observed in mice, such as reduction in the percentage of colonic stem cells, promotion of stem cell differentiation, and attenuation of FoxM1 signaling. These findings indicate that the AhR-FoxM1 axis, at least in part, mediates colonic stem/progenitor cell behavior.

Aryl Hydrocarbon Receptor Activity in Intestinal Epithelial Cells in the Formation of Colonic Tertiary Lymphoid Tissues.

After birth, the development of secondary lymphoid tissues (SLTs) in the colon is dependent on the expression of the Aryl Hydrocarbon Receptor (AhR) in immune cells as a response to the availability of AhR ligands. However, little is known about how AhR activity from intestinal epithelial cells (IECs) may influence the development of tertiary lymphoid tissues (TLTs). As organized structures that develop at sites of inflammation or infection during adulthood, TLTs serve as localized centers of adaptive immune responses, and their presence has been associated with the resolution of inflammation and tumorigenesis in the colon. Here, we investigated the effect of the conditional loss of AhR activity in IECs in the formation and immune cell composition of TLTs in a model of acute inflammation. In females, loss of AhR activity in IECs reduced the formation of TLTs without significantly changing disease outcomes nor immune cell composition within TLTs. In males lacking AhR expression in IECs, increased disease activity index, lower expression of functional-IEC genes, increased number of TLTs, increased T-cell density, and lower B- to T-cell ratio was observed. These findings may represent an unfavorable prognosis when exposed to DSS-induced epithelial damage compared to females. Sex and loss of IEC AhR also resulted in changes in microbial populations in the gut. Collectively, these data suggest that the formation of TLTs in the colon is influenced by sex and AhR expression in IECs.

Novel Role of Ghrelin Receptor in Gut Dysbiosis and Experimental Colitis in Aging.

Chronic low-grade inflammation is a hallmark of aging, which is now coined as inflamm-aging. Inflamm-aging contributes to many age-associated diseases such as obesity, type 2 diabetes, cardiovascular disease, and inflammatory bowel disease (IBD). We have shown that gut hormone ghrelin, via its receptor growth hormone secretagogue receptor (GHS-R), regulates energy metabolism and inflammation in aging. Emerging evidence suggests that gut microbiome has a critical role in intestinal immunity of the host. To determine whether microbiome is an integral driving force of GHS-R mediated immune-metabolic homeostasis in aging, we assessed the gut microbiome profiles of young and old GHS-R global knockout (KO) mice. While young GHS-R KO mice showed marginal changes in Bacteroidetes and Firmicutes, aged GHS-R KO mice exhibited reduced Bacteroidetes and increased Firmicutes, featuring a disease-susceptible microbiome profile. To further study the role of GHS-R in intestinal inflammation in aging, we induced acute colitis in young and aged GHS-R KO mice using dextran sulfate sodium (DSS). The GHS-R KO mice showed more severe disease activity scores, higher proinflammatory cytokine expression, and decreased expression of tight junction markers. These results suggest that GHS-R plays an important role in microbiome homeostasis and gut inflammation during aging; GHS-R suppression exacerbates intestinal inflammation in aging and increases vulnerability to colitis. Collectively, our finding reveals for the first time that GHS-R is an important regulator of intestinal health in aging; targeting GHS-R may present a novel therapeutic strategy for prevention/treatment of aging leaky gut and inflammatory bowel disease.

Effects of high-fat diet and intestinal aryl hydrocarbon receptor deletion on colon carcinogenesis.

Consumption of a high-fat diet has been associated with an increased risk of developing colorectal cancer (CRC). However, the effects of the interaction between dietary fat content and the aryl hydrocarbon receptor (AhR) on colorectal carcinogenesis remain unclear. Mainly known for its role in xenobiotic metabolism, AhR has been identified as an important regulator for maintaining intestinal epithelial homeostasis. Although previous research using whole body AhR knockout mice has revealed an increased incidence of colon and cecal tumors, the unique role of AhR activity in intestinal epithelial cells (IECs) and modifying effects of fat content in the diet at different stages of sporadic CRC development are yet to be elucidated. In the present study, we have examined the effects of a high-fat diet on IEC-specific AhR knockout mice in a model of sporadic CRC. Although loss of AhR activity in IECs significantly induced the development of premalignant lesions, in a separate experiment, no significant changes in colon mass incidence were observed. Moreover, consumption of a high-fat diet promoted cell proliferation in crypts at the premalignant colon cancer lesion stage and colon mass multiplicity as well as ß-catenin expression and nuclear localization in actively proliferating cells in colon masses. Our data demonstrate the modifying effects of high-fat diet and AhR deletion in IECs on tumor initiation and progression.NEW & NOTEWORTHY Through the use of an intestinal-specific aryl hydrocarbon receptor (AhR) knockout mouse model, this study demonstrates that the expression of AhR in intestinal epithelial cells is required to reduce the formation of premalignant colon cancer lesions. Furthermore, consumption of a high-fat diet and the loss of AhR in intestinal epithelial cells influences the development of colorectal cancer at various stages.

Effect of diet and intestinal AhR expression on fecal microbiome and metabolomic profiles.

BACKGROUND: Diet, loss of aryl hydrocarbon receptor (AhR) expression and their modification of the gut microbiota community composition and its metabolites affect the development of colorectal cancer (CRC). However, the concordance between fecal microbiota composition and the fecal metabolome is poorly understood. Mice with specific AhR deletion (AhRKO) in intestinal epithelial cell and their wild-type littermates were fed a low-fat diet or a high-fat diet. Shifts in the fecal microbiome and metabolome associated with diet and loss of AhR expression were assessed. Microbiome and metabolome data were integrated to identify specific microbial taxa that contributed to the observed metabolite shifts. RESULTS: Our analysis shows that diet has a more pronounced effect on mouse fecal microbiota composition than the impact of the loss of AhR. In contrast, metabolomic analysis showed that the loss of AhR in intestinal epithelial cells had a more pronounced effect on metabolite profile compared to diet. Integration analysis of microbiome and metabolome identified unclassified Clostridiales, unclassified Desulfovibrionaceae, and Akkermansia as key contributors to the synthesis and/or utilization of tryptophan metabolites. CONCLUSIONS: Akkermansia are likely to contribute to the synthesis and/or degradation of tryptophan metabolites. Our study highlights the use of multi-omic analysis to investigate the relationship between the microbiome and metabolome and identifies possible taxa that can be targeted to manipulate the microbiome for CRC treatment.

Isoflavones as Ah Receptor Agonists in Colon-Derived Cell Lines: Structure-Activity Relationships.

Many of the protective responses observed for flavonoids in the gastrointestinal track resemble aryl hydrocarbon receptor (AhR)-mediated effects. Therefore, we examined the structure-activity relationships of isoflavones and isomeric flavone and flavanones as AhR ligands on the basis of their induction of CYP1A1, CYP1B1, and UGT1A1 gene expression in colon cancer Caco2 cells and young adult mouse colonocyte (YAMC) cells. Caco2 cells were significantly more Ah-responsive than YAMC cells, and this was due, in part, to flavonoid-induced cytotoxicity in the latter cell lines. The structure-activity relationships for the flavonoids were complex and both response and cell context specific; however, there was significant variability in the AhR activities of the isomeric substituted isoflavones and flavones. For example, 4',5,7-trihydroxyisoflavone (genistein) was AhR-inactive whereas 4',5,7-trihydroxyflavone (apigenin) induced CYP1A1, CYP1B1, and UGT1A1 in Caco2 cells. In contrast, both 5,7-dihydroxy-4-methoxy substituted isoflavone (biochanin A) and flavone (acacetin) induced all three AhR-responsive genes; 4',5,7-trimethoxyisoflavone was a potent AhR agonist, and the isomeric flavone was AhR-inactive. These results coupled with simulation studies modeling flavonoid interaction within the AhR binding pocket demonstrate that the orientation of the substituted phenyl ring at C-2 (flavones) or C-3 (isoflavones) on the common 4-H-chromen-4-one ring strongly influences the activities of isoflavones and flavones as AhR agonists.

Flaxseed Bioactive Compounds and Colorectal Cancer Prevention.

PURPOSE OF REVIEW: Flaxseed and its bioactive components have been associated with a decreased risk of colorectal cancer incidence and progression. This review aims to summarize recent research regarding the role of flaxseed and each of its major dietary bioactive components in reducing colorectal cancer. RECENT FINDINGS: In both human and animal model experiments, flaxseed consumption had beneficial effects on colon physiology associated with reduction in colorectal cancer risk or occurrence. Considered separately, each of flaxseed's major bioactive components, including fiber, alpha-linolenic acid, lignans, and other phytochemicals, is also associated with decreased risk of colonic neoplasms and regulation of cell growth through several potential mechanisms. Collectively, experimental data suggests that consumption of flaxseed and/or its bioactive components may reduce colorectal cancer risk by a variety of mechanisms. Future studies should focus on the mechanisms by which whole flaxseed can prevent colorectal cancer.

Estradiol Has Differential Effects on Acute Colonic Inflammation in the Presence and Absence of Estrogen Receptor ß Expression.

BACKGROUND: Inflammatory bowel disease (IBD) increases the risk of developing colon cancer. This risk is higher in men compared to women, implicating a role for female hormones in the protection against this disease. Studies from our laboratory demonstrated that estradiol (E2) protects against inflammation-associated colon tumor formation when administered following chemical carcinogen and induction of chronic colitis. AIM: This study seeks to better understand the effect of E2 on acute colitis in the presence and absence of estrogen receptor ß (ERß). METHODS: Inflammation was induced by 2,4,6-trinitrobenzenesulfonic acid in wild-type (WT) and ERß knockout (ERßKO) mice implanted with a control or E2-containing pellet and killed 5 days later. Inflammation and injury were scored by a pathologist. Apoptosis and proliferation were assessed by immunohistochemistry. Cytokines were measured by multiplex analysis. RESULTS: E2 treatment reduced inflammation in the middle colon in WT mice and the distal colon in ERßKO mice compared to control mice. WT mice had reduced IL-6, IL-12, IL-17, GM-CSF, IFN-γ, MCP-1, MIP-1α, and TNF-α, and ERßKO had reduced IL-6 and IFN-γ expression in response to E2. Injury scores were lower in E2-treated ERßKO mice compared to control ERßKO mice. ERßKO mice had increased proliferation in the basal third of crypts in the distal colon and decreased apoptosis in the proximal colon. CONCLUSIONS: These data suggest that E2 has differential protective effects against acute colitis in the presence or absence of ERß and provide insight into how E2 may protect against IBD.

Homeostatic responses of colonic LGR5+ stem cells following acute in vivo exposure to a genotoxic carcinogen.

Perturbations in DNA damage, DNA repair, apoptosis and cell proliferation in the base of the crypt where stem cells reside are associated with colorectal cancer (CRC) initiation and progression. Although the transformation of leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)(+) cells is an extremely efficient route towards initiating small intestinal adenomas, the role of Lgr5(+) cells in CRC pathogenesis has not been well investigated. Therefore, we further characterized the properties of colonic Lgr5(+) cells compared to differentiated cells in Lgr5-EGFP-IRES-creER(T2) knock-in mice at the initiation stage of carcinogen azoxymethane (AOM)-induced tumorigenesis using a quantitative immunofluorescence microscopy approach. At 12 and 24h post-AOM treatment, colonic Lgr5(+) stem cells (GFP(high)) were preferentially damaged by carcinogen, exhibiting a 4.7-fold induction of apoptosis compared to differentiated (GFP(neg)) cells. Furthermore, with respect to DNA repair, O(6)-methylguanine DNA methyltransferase (MGMT) expression was preferentially induced (by 18.5-fold) in GFP(high) cells at 24h post-AOM treatment compared to GFP(neg) differentiated cells. This corresponded with a 4.3-fold increase in cell proliferation in GFP(high) cells. These data suggest that Lgr5(+) stem cells uniquely respond to alkylation-induced DNA damage by upregulating DNA damage repair, apoptosis and cell proliferation compared to differentiated cells in order to maintain genomic integrity. These findings highlight the mechanisms by which colonic Lgr5(+) stem cells respond to cancer-causing environmental factors.

Diet complexity and estrogen receptor ß status affect the composition of the murine intestinal microbiota.

Intestinal microbial dysbiosis contributes to the dysmetabolism of luminal factors, including steroid hormones (sterones) that affect the development of chronic gastrointestinal inflammation and the incidence of sterone-responsive cancers of the breast, prostate, and colon. Little is known, however, about the role of specific host sterone nucleoreceptors, including estrogen receptor ß (ERß), in microbiota maintenance. Herein, we test the hypothesis that ERß status affects microbiota composition and determine if such compositionally distinct microbiota respond differently to changes in diet complexity that favor Proteobacteria enrichment. To this end, conventionally raised female ERß(+/+) and ERß(-/-) C57BL/6J mice (mean age of 27 weeks) were initially reared on 8604, a complex diet containing estrogenic isoflavones, and then fed AIN-76, an isoflavone-free semisynthetic diet, for 2 weeks. 16S rRNA gene surveys revealed that the fecal microbiota of 8604-fed mice and AIN-76-fed mice differed, as expected. The relative diversity of Proteobacteria, especially the Alphaproteobacteria and Gammaproteobacteria, increased significantly following the transition to AIN-76. Distinct patterns for beneficial Lactobacillales were exclusive to and highly abundant among 8604-fed mice, whereas several Proteobacteria were exclusive to AIN-76-fed mice. Interestingly, representative orders of the phyla Proteobacteria, Bacteroidetes, and Firmicutes, including the Lactobacillales, also differed as a function of murine ERß status. Overall, these interactions suggest that sterone nucleoreceptor status and diet complexity may play important roles in microbiota maintenance. Furthermore, we envision that this model for gastrointestinal dysbiosis may be used to identify novel probiotics, prebiotics, nutritional strategies, and pharmaceuticals for the prevention and resolution of Proteobacteria-rich dysbiosis.

Aryl Hydrocarbon Receptor (AhR) Signaling in Colonic Cells and Tumors.

The aryl hydrocarbon receptor (AhR) is overexpressed in many tumor types and exhibits tumor-specific tumor promoter and tumor suppressor-like activity. In colon cancer, most but not all studies suggest that the AhR exhibits tumor suppressor activity which is enhanced by AhR ligands acting as agonists. Our studies investigated the role of the AhR in colon tumorigenesis using wild-type and AhR-knockout mice, the inflammation model of colon tumorigenesis using mice treated with azoxymethane (AOM)/dextran sodium sulfate (DSS) and APCS580/+; KrasG12D/+ mice all of which form intestinal tumors. The effects of tissue-specific AhR loss in the intestine of the tumor-forming mice on colonic stem cells, organoid-initiating capacity, colon tumor formation and mechanisms of AhR-mediated effects were investigated. Loss of AhR enhanced stem cell and tumor growth and in the AOM/DSS model AhR-dependent suppression of FOXM1 and downstream genes was important for AhR-dependent anticancer activity. Furthermore, the effectiveness of interleukin-22 (IL22) in colonic epithelial cells was also dependent on AhR expression. IL22 induced phosphorylation of STAT3, inhibited colonic organoid growth, promoted colonic cell proliferation in vivo and enhanced DNA repair in AOM/DSS-induced tumors. In this mouse model, the AhR suppressed SOCS3 expression and enhanced IL22-mediated activation of STAT3, whereas the loss of the AhR increased levels of SOCS3 which in turn inhibited IL22-induced STAT3 activation. In the APCS580/+; KrasG12D/+ mouse model, the loss of the AhR enhanced Wnt signaling and colon carcinogenesis. Results in both mouse models of colon carcinogenesis were complemented by single cell transcriptomics on colonic intestinal crypts which also showed that AhR deletion promoted expression of FOXM1-regulated genes in multiple colonic cell subtypes. These results support the role of the AhR as a tumor suppressor-like gene in the colon.

Sorghum phenolics demonstrate estrogenic action and induce apoptosis in nonmalignant colonocytes.

Evidence indicates sorghum may be protective against colon cancer; however, the mechanisms are unknown. Estrogen is believed to protect against colon cancer development by inducing apoptosis in damaged nonmalignant colonocytes. Three sorghum extracts (white, red, and black) were screened for estrogenic activity using cell models expressing estrogen receptor α (ER-α; MCF-7 breast cancer cells) and ß [ER-ß; nonmalignant young adult mouse colonocytes (YAMC)]. Black and white sorghum extracts had significant estrogenic activity mediated through both estrogen receptors at 1-5 and 5-10 µg/mL, respectively; but red sorghum did not. Activation of ER-ß in YAMC reduced cell growth via induction of apoptosis. Only the black and red sorghums contained 3-deoxyanthocyanins; however, these compounds were non-estrogenic. Flavones with estrogenic properties, luteolin (0.41-2.12 mg/g) and apigenin (1.1-1.4 mg/g), and their O-methyl derivatives (0.70-0.95 mg/g) were detected in white and black sorghums, but not in the red sorghum. On the other hand, naringenin, a flavanone known to interfere with transcriptional activities of estrogen, was only detected in the red sorghum extract (as its 7-O-glycoside) at relatively high concentration (11.8 mg/g). Sorghum flavonoid composition has important implications on possible modes of chemoprotection by sorghum against colon carcinogenesis.

Dietary fish oil reduces DNA adduct formation while estradiol upregulates apoptosis in response to DNA damage in the rat colon.

BACKGROUND: Dietary fish oil is associated with a decrease in colon cancer incidence: in part through a reduction in DNA adduct formation and an induction of colonocyte apoptosis. Estradiol (E(2)) has also been demonstrated to be protective against colon cancer incidence. Studies evaluating fish oil diets and DNA adduct formation in the colon have been conducted in male models without regard to possible interactions with E(2). AIMS: The aim of this study was to evaluate the effects of E(2) and fish oil both together and separately in female rats at the point of DNA damage. METHODS: Ovariectomized female Sprague-Dawley rats were fed either a corn oil or fish oil diet in the presence or absence of E(2) for two weeks prior to being sacrificed at four time points following injection with azoxymethane. O(6)-methyldeoxyguanosine (O(6)-MedG) DNA adducts and apoptosis were examined using immunohistochemistry. RESULTS: Dietary fish oil reduced DNA adduct formation independent of the presence of E(2) at both 9 and 12 h post carcinogen. E(2) itself did not suppress adduct formation. E(2) significantly induced apoptosis 12 h after carcinogen independent of diet, primarily in the luminal third of the crypts. Fish oil was not associated with increased colonocyte apoptosis. CONCLUSIONS: These data demonstrate that fish oil is protective against DNA damage in the colon regardless of gender through reduction of O(6)-MedG adduct formation. Additionally, E(2) is capable of inducing apoptosis directly at the point of DNA damage.

Estrogen receptor actions in colitis.

In recent years, researchers have demonstrated that estrogen and its receptors, aside from their role in regulating several biological functions, contribute to the development and progression/severity of inflammatory bowel diseases (IBDs). IBDs include both ulcerative colitis (UC) and Crohn's disease (CD). Epidemiological data indicate a clear difference in the incidence, severity, and complications of IBDs between sexes. Men present a higher risk of developing colitis than women and a higher risk of developing colorectal cancer, a common complication of this condition. However, fluctuations of estrogen levels have yielded inconsistent data, where oral contraceptives and hormone replacement therapy have been associated with an increased risk of IBDs in premenopausal women but significantly reduce disease activity after menopause. Likewise, improvement of symptoms related to CD has been reported during pregnancy, but not in UC, who often experience worsening symptoms. In the colonic epithelium, estrogen receptor ß (ERß) is the predominant form of the protein expressed, and it helps maintain normal epithelial function and organization. Preclinical data suggest that ER expression and activation via estrogen confers different responses on disease severity depending on the model used to induce colitis, which may reflect what is observed in patients with IBDs. Hence, this review aims to provide an overview of estrogen and its receptors, particularly ERß, in the pathophysiology of IBDs.

Trigonelline is a novel phytoestrogen in coffee beans.

Drinking coffee has been associated with the development of several endocrine-related cancers. The interpretation of these data has often been limited to the role that caffeine plays. Trigonelline (Trig), a niacin-related compound, is a natural constituent of coffee accounting for approximately 1% dry matter in roasted beans. Studies exploring the effects of this bioactive compound on mammalian cells are limited. The initial purpose of our studies was to determine whether Trig alters the actions of estradiol (E(2)), using proliferation of estrogen-dependent human breast cancer (MCF-7) cells as a model system. When cells were cotreated with suboptimal doses of E(2) (10 pmol/L) and Trig (100 pmol/L), an additive enhancement of MCF-7 growth was observed. In the absence of E(2), Trig stimulated MCF-7 cell proliferation in a dose-responsive manner and significantly enhanced cell growth at concentrations as low as 100 pmol/L. Cotreatment of MCF-7 cells with Trig and ICI 182,780, an estrogen receptor (ER) antagonist, inhibited Trig-induced cell proliferation. Trig treatment also induced activation of estrogen response element reporter assays in MCF-7 cells and increased expression of ER target genes (pS2, progesterone receptor, and cyclin D1) similar to E(2). While our data demonstrate that Trig activates the ER, competitive binding assays showed that Trig does not compete E(2) off of the ER at any concentration. This suggests that Trig is activating the ER through a separate mechanism. Collectively, these data demonstrate that Trig even at low concentrations stimulates MCF-7 cell growth and that this effect is mediated through ER, clearly identifying Trig as a novel phytoestrogen.

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice.

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: α cells secrete glucagon, ß cells secrete insulin, δ cells secrete somatostatin, ε cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are ß cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250-350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

PPARgamma1 as a molecular target of eicosapentaenoic acid in human colon cancer (HT-29) cells.

Diets high in (n-3) PUFA decrease colon cancer development and suppress colon tumor growth, but the molecular mechanism through which these compounds act is largely unknown. We sought to determine whether PPARgamma1 serves as a molecular link between the physiological actions of eicosapentaenoic acid (EPA) in human colon cancer cells (HT-29). At nutritionally relevant concentrations, EPA stimulated a PPAR response element (PPRE) reporter assay in a dose-responsive manner in HT-29 cells. Cotreatment with GW9662 (GW), a PPARgamma antagonist, significantly inhibited this effect, whereas overexpressing the receptor enhanced it. EPA also stimulated the PPRE reporter in a PPARgamma negative cancer cell line (22Rv1) when the cells were cotransfected with a PPARgamma1 expression plasmid and this effect was again inhibited by GW. Furthermore, in vitro incubation of EPA with PPARgamma1 enhanced binding of the protein to DNA containing a PPRE. Next, we sought to determine whether EPA or a prostaglandin formed from EPA is the functional ligand of PPARgamma. Cotreatment in HT-29 and 22Rv1 cells with EPA and acetyl salicylic acid, an inhibitor of cyclooxygenase activity, activated the PPRE reporter at levels similar to EPA alone, suggesting that EPA itself is a ligand of PPARgamma. Finally, EPA suppressed HT-29 cell growth and this effect was significantly reversed by the addition of GW, suggesting that in part the physiological actions of EPA are the result of PPARgamma activation. These studies identify PPARgamma as a molecular mediator of (n-3) PUFA actions in colon cancer cells.

Effects of chronic estradiol treatment on delayed spatial alternation and differential reinforcement of low rates of responding.

Estrogens have been shown to both enhance and impair cognitive function depending on several factors, including regimen of hormone treatment, age of subject, and task attributes. In rodent models, estradiol tends to enhance spatial learning and impair response or cued learning, but effects on executive functions are less well-studied. In this experiment, spatial working memory and response inhibition were tested using delayed spatial alternation (DSA) and differential reinforcement of low rates of responding (DRL) tasks in ovariectomized rats that were given chronic estradiol via Silastic implants resulting in serum estradiol concentrations of 86.2 +/- 8.2 (SEM) pg/ml. Rats were tested for 25 days DSA with variable delays of 0, 3, 6, 9, and 18 seconds between lever presentations, followed by 30 days on a DRL-15s operant schedule. Estradiol-replaced rats showed a significantly lower proportion of correct responses on the DSA task compared to vehicle-implanted ovariectomized animals. On DRL, estradiol-treated rats showed a lower ratio of reinforced to nonreinforced presses. These data suggest that chronic estrogen exposure may impair rats' abilities on measures of executive function including working memory and response inhibition.

Bisphenol-A alters microbiota metabolites derived from aromatic amino acids and worsens disease activity during colitis.

Inflammatory bowel disease is a complex collection of disorders. Microbial dysbiosis as well as exposure to toxins including xenoestrogens are thought to be risk factors for inflammatory bowel disease development and relapse. Bisphenol-A has been shown to exert estrogenic activity in the colon and alter intestinal function, but the role that xenoestrogens, such as bisphenol-A , play in colonic inflammation has been previously described but with conflicting results. We investigated the ability of bisphenol-A to exacerbate colonic inflammation and alter microbiota metabolites derived from aromatic amino acids in an acute dextran sulfate sodium-induced colitis model. Female C57BL/6 mice were ovariectomized and exposed to bisphenol-A daily for 15 days. Disease activity measures include body weight, fecal consistency, and rectal bleeding. Colons were scored for inflammation, injury, and nodularity. Alterations in the levels of microbiota metabolites derived from aromatic amino acids known to reflect phenotypic changes in the gut microbiome were analyzed. Bisphenol-A exposure increased mortality and worsened disease activity as well as inflammation and nodularity scores in the middle colon region following dextran sulfate sodium exposure. Unique patterns of metabolites were associated with bisphenol-A consumption. Regardless of dextran sulfate sodium treatment, bisphenol-A reduced levels of tryptophan and several metabolites associated with decreased inflammation in the colon. This is the first study to show that bisphenol-A treatment alone can reduce microbiota metabolites derived from aromatic amino acids in the colon which may be associated with increased colonic inflammation and inflammatory bowel disease. Impact statement As rates of inflammatory bowel disease rise, discovery of the mechanisms related to the development of these conditions is important. Environmental exposure is hypothesized to play a role in etiology of the disease, as are alterations in the gut microbiome and the metabolites they produce. This study is the first to show that bisphenol-A alone alters tryptophan and microbiota metabolites derived from aromatic amino acids in a manner consistent with autoimmune diseases, specifically inflammatory bowel diseases, regardless of dextran sulfate sodium treatment. These findings indicate a potential mechanism by which bisphenol-A negatively affects gut physiology to exacerbate inflammation.

Dietary genistein negates the inhibitory effect of tamoxifen on growth of estrogen-dependent human breast cancer (MCF-7) cells implanted in athymic mice.

The use of dietary isoflavone supplements by postmenopausal women with breast cancer is increasing. We investigated interactions between the soy isoflavone, genistein, and an antiestrogen, tamoxifen (TAM), on the growth of estrogen (E)-dependent breast cancer (MCF-7) cells implanted in ovariectomized athymic mice. We hypothesized that weakly estrogenic genistein negate/overwhelm the inhibitory effect of TAM on the growth of E-dependent breast tumors. Six treatment groups were used: control (C); 0.25 mg estradiol (E2) implant (E); E2 implant + 2.5 mg TAM implant (2.5 TE); E2 implant + 2.5 mg TAM implant + 1000 ppm genistein (2.5 TEG); E2 implant + 5 mg TAM implant (5 TE), and E2 implant +5 mg TAM implant +1000 ppm genistein (5 TEG). Treatment with TAM (2.5 TE and 5 TE) suppressed E2-stimulated MCF-7 tumor growth in ovariectomized athymic mice. Dietary genistein negated/overwhelmed the inhibitory effect of TAM on MCF-7 tumor growth, lowered E2 level in plasma, and increased expression of E-responsive genes (e.g., pS2, PR, and cyclin D1). Therefore, caution is warranted for postmenopausal women consuming dietary genistein while on TAM therapy for E-responsive breast cancer.