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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 or 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 were observed. These findings may represent an unfavorable prognosis when exposed to dextran sodium sulfate (DSS)-induced epithelial damage compared with 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. NEW & NOTEWORTHY This is the first research of its kind to demonstrate a clear connection between biological sex and the development of tertiary lymphoid tissues (TLT) in the colon. In addition, the research finds that in a preclinical model of inflammatory bowel disease, the expression of the aryl hydrocarbon receptor (AhR) influences the development of these structures in a sex-specific manner.

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., (© 2020 The Authors.)

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 β-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.

Epidemiological and animal data have demonstrated the protective effects of estrogen signaling on colon carcinogenesis. Nonetheless, studies have suggested that estrogen replacement therapy is positively correlated to increased risk of breast cancer. Therefore, there is considerable interest in investigating novel phytoestrogens that mimic the protective actions of estrogen in the colon. Trigonelline (Trig) and 3,3-diindolylmethane (DIM) have been reported as phytoestrogens in spite of their distinct chemical structures from other phytoestrogens. Both compounds elicit estrogenic responses without directly interacting with the binding domain of the estrogen receptor (ER). We examined the influence of Trig and DIM on non-malignant colonocytes. Both compounds reduced cell growth of young adult mouse colonocytes (YAMCs). Trig and DIM induced cell cycle arrest in the G0/G1 phase and enhanced apoptosis in YAMCs. The inhibitory effect of Trig on cell growth was disrupted by co-treatment of ICI 182,780, an ER antagonist. DIM elevated ER mediated transcriptional activity. Both compounds changed gene expression related to apoptosis and cell proliferation in unique ways. In conclusion, Trig and DIM impact cell physiology and gene expression in YAMCs via novel estrogenic actions and these data suggest that intake of novel phytoestrogens may activate protective effects of estrogen signaling in the colon., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The tryptophan microbiota metabolites indole-3-acetate, indole-3-aldehyde, indole, and tryptamine are aryl hydrocarbon receptor (AhR) ligands, and in this study we investigated their AhR agonist and antagonist activities in nontransformed young adult mouse colonocyte (YAMC) cells. Using Cyp1a1 mRNA as an Ah-responsive end point, we observed that the tryptophan metabolites were weak AhR agonists and partial antagonists in YAMC cells, and the pattern of activity was different from that previously observed in CaCo2 colon cancer cells. However, expansion of the end points to other Ah-responsive genes including the Cyp1b1, the AhR repressor (Ahrr), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP-ribose) polymerase (TiParp) revealed a highly complex pattern of AhR agonist/antagonist activities that were both ligand- and gene-dependent. For example, the magnitude of induction of Cyp1b1 mRNA was similar for TCDD, tryptamine, and indole-3-acetate, whereas lower induction was observed for indole and indole-3-aldehyde was inactive. These results suggest that the tryptophan metabolites identified in microbiota are selective AhR modulators., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Activation of estrogen receptor-β (ERβ) is an important mechanism for colon cancer prevention. Specific sorghum varieties that contain flavones were shown to activate ER in non-malignant colonocytes at low concentrations. This study aimed to determine positive interactions among estrogenic flavonoids most relevant in sorghum. Apigenin and naringenin were tested separately and in combination for their ability to influence ER-mediated cell growth in non-malignant young adult mouse colonocytes (YAMC). Sorghum extracts high in specific flavanones and flavones were also tested. Apigenin reduced ER-mediated YAMC cell growth comparable to physiological levels of estradiol (E₂, 1 nM) at 1 μM; naringenin had similar effect at 10 μM. However, when combined, 0.1 μM apigenin plus 0.05 μM naringenin produced similar effect as 1 nM E₂; these concentrations represented 1/10th and 1/200th, respectively, of the active concentrations of apigenin and naringenin, demonstrating a strong enhanced action. A sorghum extract higher in flavones (apigenin and luteolin) (4.8 mg g(-1)) was more effective (5 μg mL(-1)) at activating ER in YAMC than a higher flavanone (naringenin and eriodictyol) (28.1 mg g(-1)) sorghum extract (10 μg mL(-1)). Enhanced actions observed for apigenin and naringenin were adequate to explain the level of effects produced by the high flavone and flavanone sorghum extracts. Strong positive interactions among sorghum flavonoids may enhance their ability to contribute to colon cancer prevention beyond what can be modeled using target compounds in isolation.

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 β [ER-β; 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-β 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.

Clinical and animal studies have shown a strong link between estrogen status in women and decreased risk of colon cancer. However, little research has been done into the mechanism of protection that estrogen provides. Our laboratory has demonstrated that estradiol (E₂) inhibits the development of pre-neoplastic lesions through an estrogen receptor β (ERβ) mediated mechanism in mice. Our data also suggest that the primary protective role of E₂ treatment is increased apoptosis in non-malignant colonocytes that are damaged and at risk of becoming cancerous. The p53 protein plays a crucial role in the cellular response to stress by inducing cell cycle arrest, DNA repair mechanisms, and/or apoptosis. Due to the observed induction of apoptosis in response to E₂, we are investigating the role of p53 in this chemo-protective mechanism. E₂ suppressed growth of young adult mouse colonocytes (YAMCs) by inducing apoptosis and these physiological responses were completely lost in YAMCs lacking a functional p53 protein. Western blot analysis demonstrated increases in p53 protein levels in YAMCs after treatment with E₂ likely due to protein stabilization. E₂ was shown to enhance the transcriptional activity of p53, resulting in up-regulation of pro-apoptotic p53 target genes (Bax, Noxa, and PUMA). Finally, repair of DNA double stranded breaks was shown to be increased by E₂ treatment. Collectively, these data are the first to demonstrate that p53 is a primary mediator of the protective actions of E₂ in the colon., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

Numerous clinical and animal studies show that hormone replacement therapy reduces the risk of colon tumor formation. However, the majority of experiments have shown that estradiol (E(2)) does not inhibit the growth of malignantly transformed colon epithelia. As such, the presented studies focused on evaluating the effects of E(2) in noncancerous colonocytes. E(2) treatments (0-10 nmol/L) reduced cell growth and increased apoptotic activity in young adult mouse colonocytes (YAMC), a nonmalignant cell line, in a dose-responsive manner. These effects were lost in the YAMC-Ras cells, an isogenic cell line with a single malignant transformation. Cotreatment with an estrogen receptor (ER) antagonist inhibited the physiologic effects of E(2) in YAMC cells, suggesting that the response is ER mediated. To further study the effect of E(2) on colonic epithelia, we evaluated the development of preneoplastic lesions in ovariectomized wild-type (WT) and ERbeta knockout (ERbetaKO) mice treated with either vehicle or E(2). WT E(2)-treated animals exhibited significantly fewer aberrant crypt foci and increased apoptotic activity in colonic epithelia when compared with WT control mice or ERbetaKO animals receiving either treatment. For the first time, we showed that E(2) alters the growth of nontransformed colonocytes in vitro and that, through an ERbeta-mediated mechanism, E(2) influences the physiology of noncancerous colonocytes, resulting in fewer preneoplastic lesions. Collectively, these data show that the protective actions of E(2) occur primarily during the initiation/promotion stages of disease development and identify the hormone as an important chemoprotective agent.