5-O-Methylgenistein of Phaleria macrocarpa (Scheff.) Boerl. revealed as a potential therapeutic compound for endometriosis

Context: Endometriosis is a condition marked by the presence of endometrial epithelium and stromal cells outside the uterus. Previous studies have shown that prostaglandin E2 has a role in the development of endometriosis via the action of local estrogen and estrogen receptors. Aims: To analyze the molecular docking between Phaleria macrocarpa flavonoid compounds in the NF-B, VEGF/VEGFR2, Ki67, COX-2, and CXCR4 pathways that are involved in the pathomechanism of endometriosis. Methods: Functional determination of Phaleria macrocarpa phytochemistry using PASS prediction ADMETSAR's prediction of Phaleria macrocarpa's flavonoids compounds meet the Lipinski rule criteria so that they are predicted to have drug-likeness and no toxicity. Phaleria macrocarpa flavonoids were synthesized using AutoDock Tools 1.5.7 software. AutoDock Vina v1.2.3 software was used to perform docking simulations of ligands and target proteins. The results of the docking analysis were visualized with the Discovery Studio 4.1 application. Results: For the NF-B pathway, the compounds that showed the highest affinity for interaction to occur were (±)-naringenin (target of NF-B p50/p65) and (-)-8-prenylnaringenin (the ATP-binding site in IKK). Against VEGF-A and VEGFR2, the flavonoids that exhibited the highest interaction affinity were glycitin, and (-)-8-prenylnaringenin, respectively. The compound that interacted most easily with Ki67, COX-2, or CXCR4 was (+)-catechin 7-O-beta-D-xyloside. For docking with standard drugs, we found that leuprolide acetate and dienogest showed higher affinity for NF-B p50/p65 heterodimer than flavonoids. Conclusions: The six compounds Phaleria macrocarpa have different affinities for selected pathways in endometriosis. Thus, the Phaleria macrocarpa flavonoids could be a multi-action herbal candidate for endometriosis, which can be used alone or as a complement to standard drugs.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin. However, the role of the AHR in normal physiology is still an area of intense investigation. For example, this receptor plays an important role in certain immune responses. We have previously determined that the AHR can mediate repression of acute-phase genes in the liver. For this observation to be therapeutically useful, selective activation of the AHR would likely be necessary. Recently, the selective estrogen receptor ligand WAY-169916 has also been shown to be a selective AHR ligand. WAY-169916 can efficiently repress cytokine-mediated acute-phase gene expression (e.g., SAA1) yet fail to mediate a dioxin response element-driven increase in transcriptional activity. The goals of this study were to structurally modify WAY-169916 to block binding to the estrogen receptor and increase its affinity for the AHR. A number of WAY-169916 derivatives were synthesized and subjected to characterization as AHR ligands. The substitution of a key hydroxy group for a methoxy group ablates binding to the estrogen receptor and increases its affinity for the AHR. The compound 1-allyl-7-trifluoromethyl-1H-indazol-3-yl]-4-methoxyphenol (SGA 360), in particular, exhibited essentially no AHR agonist activity yet was able to repress cytokine-mediated SAA1 gene expression in Huh7 cells. SGA 360 was tested in a 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated ear inflammatory edema model using C57BL6/J and Ahr(-/-) mice. Our findings indicate that SGA 360 significantly inhibits TPA-mediated ear swelling and induction of a number of inflammatory genes (e.g., Saa3, Cox2, and Il6) in C57BL6/J mice. In contrast, SGA 360 had no effect on TPA-mediated ear swelling or inflammatory gene expression in Ahr(-/-) mice. Collectively, these results indicate that SGA 360 is a selective Ah receptor modulator (SAhRM) that exhibits anti-inflammatory properties in vivo.

Introduction: Markers for treatment resistance in breast cancer are needed. The Aryl hydrocarbon receptor (AhR) is involved in the regulation of estrogen metabolism. Previous studies have observed a crosstalk between AhR and the estrogen receptor (ER), indicating that the AhR may be of importance in the response of endocrine treatment. Materials and methods: A functional polymorphism in the AhR Arg554Lys (G>A) was analyzed in a cohort of 634 breast cancer patients included at their preoperative visits in Lund, Sweden between 2002 and 2008. AhR genotypes were studied in relation to ER status and risk for breast cancer events. Results: The frequencies of the AhR GG/ GA/ AA genotypes were 82.0%, 16.7%, and 1.3%, respectively. There was a trend towards increasing frequency of ER+ tumors with increasing number of A-alleles (P-trend = 0.03). Since few patients had the A/A genotype, patients with the G/A and A/A genotypes were combined in the survival analyses of the 576 patients with invasive tumors, no preoperative treatment, and no events detected on the postoperative metastases screen. Overall, AhR was not associated with event-free survival (LogRank P = 0.22). Among patients ever treated with chemotherapy or radiotherapy, AhR was not associated with event-free survival (LogRank P = 0.24 and P = 0.18, respectively). However, among tamoxifen-treated patients with ER+ tumors, AhR G/G carriers had a significantly lower risk for breast cancer events (LogRank P = 0.005), adjusted HR 0.53 (95% CI 0.29-0.94). This association was confined to patients who had received both tamoxifen and aromatase inhibitor (AI) switch treatment (LogRank P = 0.0002), adjusted HR 0.23 (0.09-055), while no association was seen in patients treated with tamoxifen only (LogRank P = 0.56). AI-treated patients with the G/G genotype had an even lower risk for breast cancer events than tamoxifen-treated patients (LogRank P = 0.005), adjusted HR 0.39 (0.19-0.81). Again this was only seen in patients who had received both AI and tamoxifen, but not in patients who had received AI but no tamoxifen treatment. Adjustments were made for age at inclusion, axillary lymph node involvement, invasive tumor size, and histological grade. Further adjustment for smoking status did not materially change the results. Conclusion: The functional AhR Arg554Lys polymorphism may impact tumor ER expression and response to endocrine switch treatment with both AI and tamoxifen in breast cancer patients. Citation Format: Maria Simonsson, Andrea Markkula, Carsten Rose, Christian Ingvar, Helena C. Jernström. AhR Arg554Lys impacts tumor ER expression and endocrine treatment response in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-194.

Evidence for ligand-mediated selective modulation of aryl hydrocarbon receptor activity.

A CCAAT/Enhancer-binding Protein Site within Antioxidant/Electrophile Response Element Along with CREB-binding Protein Participate in the Negative Regulation of RatGST-Ya Gene in Vascular Smooth Muscle Cells

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor best known for mediating xenobiotic-induced toxicity. AhR requires aryl hydrocarbon receptor nuclear translocator (ARNT) to form an active transcription complex and promote the activation of genes which have dioxin responsive element in their regulatory regions. The present study was performed to determine the complete cDNA sequences of porcine AhR and ARNT genes and their chromosomal localization. Total RNA from porcine livers were used to obtain the sequence of the entire porcine transcriptome by next-generation sequencing (NGS; lllumina HiSeq2500). In addition, both, in silico analysis and fluorescence in situ hybridization (FISH) were used to determine chromosomal localization of porcine AhR and ARNT genes. In silico analysis of nucleotide sequences showed that there were two transcript variants of AhR and ARNT genes in the pig. In addition, computer analysis revealed that AhR gene in the pig is located on chromosome 9 and ARNT on chromosome 4. The results of FISH experiment confirmed the localization of porcine AhR and ARNT genes. In the present study, for the first time, the full cDNAs of AhR and ARNT were demonstrated in the pig. In future, it would be interesting to determine the tissue distribution of AhR and ARNT transcript variants in the pig and to test whether these variants are associated with different biological functions and/or different activation pathways.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that binds environmental toxicants and regulates gene expression. AHR also regulates developmental processes, like craniofacial development and hematopoiesis, in the absence of environmental exposures. Zebrafish have 3 paralogs of AHR: ahr1a, ahr1b, and ahr2. Adult zebrafish with mutations in ahr2 exhibited craniofacial and fin defects. However, the degree to which ahr1a and ahr1b influence ahr2 signaling and contribute to fin and craniofacial development are not known. We compared morphology of adult ahr2 mutants and ahr1a;ahr1b single and double mutant zebrafish. We found that ahr1a;ahr1b single and double mutants were morphologically normal whereas ahr2 mutant zebrafish demonstrated fin and craniofacial malformations. At 5 days post fertilization, both ahr1a;ahr1b and ahr2 mutant larvae were normal, suggesting that adult phenotypes are due to defects in maturation or maintenance. Next, we analyzed the function of zebrafish AHRs activated by environmental ligands. The prototypical AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induces toxicity in humans and rodents via AHR and causes cardiotoxicity in zebrafish embryos. It has been shown that embryos with mutations in ahr2 are resistant to TCDD toxicity, yet it is unclear whether ahr1 receptors are required. Furthermore, though AHR was shown to interact with estrogen receptor alpha following TCDD treatment, it is not known whether this interaction is constitutive or context-dependent. To determine whether estrogen receptors are constitutive cofactors for AHR signaling, we used genetic and pharmacologic techniques to analyze TCDD-dependent toxicity in estrogen receptor and ahr mutant embryos. We found that embryos with mutations in ahr1a;ahr1b or estrogen receptor genes are susceptible to TCDD toxicity whereas ahr2 mutant embryos are TCDD-resistant. Moreover, pharmacologic blockade of nuclear estrogen receptors failed to prevent TCDD toxicity. These findings suggest that ahr1 genes do not have overlapping functions with ahr2 in fin and craniofacial development or TCDD-dependent toxicity, and that estrogen receptors are not constitutive partners of ahr2.

Objective To explore the relationship between aryl hydrocar-bon receptor (AhR), aryl hydrocarbon receptor nuclear translocator (ARNT), estradiol (E2), estrogen receptor (ER) and recurrent spontaneous abortion (RSA) through observing the level of serum AhR, ARNT, E2, and AhR, ARNT, ER in decidua and chorionic tissues of the patients with recurrent spontaneous abortion. Methods 64 cases of RSA patients who induced abortion at the Shanxi Dayi hospital from May 2015 to September 2017 were chosen as RSA group, and 30 cases of healthy abortion women of the same period who had born full-term normal fetus were choosen as the normal group. The serum, villi and decidua of each case were collected during abortion. The level of AhR, ARNT, ER of both groups were detected by enzyme-linked immunosorbent assay (ELISA) method. The Serum E2 lever were detected by the method of chemical luminescence. The data of all the patients were analyzed. Results ⑴ The level of ARNT in peripheral blood of RSA group was significantly higher than that of normal group (P 0.05). The levels of AhR and ARNT in villi and decidua were significantly higher in RSA group than in normal group (P 0.05); the ratio of AhR/ER and ARNT/ER in the villi and decidua of the RSA group was higher than that of the normal group (P<0.05). Conclusions Overexpression of AhR and ARNT and low expression of ER in villi and decidua tissues may be related to the occurrence of RSA. Key words: Abortion, habitual/ME; Receptors, aryl hydrocarbon/ME; Aryl hydrocarbon receptor nuclear translocator/ME; Estradiol/ME; Receptors, estrogen/ME

Chapter One - AHR- and ER-Mediated Toxicology and Chemoprevention

The aryl hydrocarbon receptor (AhR) is a key protein involved in numerous metabolic processes and signaling pathways across various cell types. Part of the basic helix-loop-helix (bHLH) transcription factor family, AhR contains a central PER-ARNT-SIM (PAS) domain crucial for binding ligands. When AhR binds to xenobiotic (toxic) substances, it becomes activated and influences a range of biological functions, such as cell proliferation, apoptosis, adhesion, differentiation, and the regulation of other transcription factors. However, the binding of certain ligands can also trigger harmful effects, contributing to disease development. Given that AhR serves as a link between the body and the external environment, understanding its response to environmental toxins, xenobiotics, and carcinogens is vital. Yet, studying AhR’s role in toxicology, physiology, and disease is challenging due to limited structural data. To overcome this, computational molecular modeling techniques like molecular docking, molecular dynamics (MD) simulations, and homology modeling have become crucial tools in AhR research. These methods provide valuable insights into how AhR is activated, its modulatory effects, and help complement experimental studies. This review highlights the use of in silico approaches to better understand AhR activation, its role in biological activities, disease progression, and potential therapeutic applications.

The activation mechanism of estrogen receptor (ER) signaling by association with the aryl hydrocarbon receptor (AhR) was elucidated recently (Ohtake, et al., Nature 2003, 423, 545). In the present study, we established a reporter yeast strain to evaluate this ER signaling by association with the activated AhR. This yeast strain expresses human ER and AhR, and has a reporter plasmid with estrogen response elements. With this yeast strain we assayed ER activation by various AhR ligands, i.e., 2,3,7,8-tetrachlorodibenzo-p-dioxin, benzo[a]pyrene, 3-methylcholanthrene, beta-naphthoflavone, and indirubin. All these ligands induced ER activation dose-dependently and their EC50 values were 60, 180, 130, 26, and 0.5 nM, respectively. Then, we measured the activity in water collected at 5 localities in the Ishizu River system in Japan. The activities of water samples ranged from 4.8 pmol/L (1.3 ng/L) to 52 pmol/L (14 ng/ L) (17beta-estradiol (E2) equivalent). These values were higher than those measured with the yeast for ER activation through direct ligand binding to ER. The direct ER ligand binding activities of the water samples ranged from 2.5 to 5.3 pmol/L (E2 equivalent). We also measured AhR activation of the water samples using a reporter yeast for AhR ligand activity. The activities ranged from 102 to 472 pmol/L (beta-naphthoflavone equivalent). These results indicate that the water samples contain substances that bind to AhR, and these substances contribute to ER signaling through AhR activation in the yeast reporter strain. This yeast reporter strain should be a useful tool to evaluate direct and indirect ER activation by environmental samples.

Estrogen receptors (ERs) and the aryl hydrocarbon receptor (AhR) are ligand activated transcription factors and members of the nuclear receptor and bHLH-PAS superfamilies, respectively. AhR is involved in xenobiotic metabolism and in mediating the toxic effects of dioxin-like compounds. Crosstalk has been observed among AhR and nuclear receptors, but has been most well studied with respect to ER signaling. Activated AhR inhibits ER activity through a number of different mechanisms, whereas ERα has been reported to have a positive role in AhR signaling. Here we will discuss recent data revealing that dioxin bound AhR recruits ERα to AhR regulated genes. We will also consider the implications of ER recruitment to AhR target genes on ER and AhR signaling.

The aryl hydrocarbon receptor (AHR) is known to mediate the cellular response to numerous xenobiotics including dioxin. Surprisingly AHR knockout mice provide evidence for the involvement of the AHR signalling cascade in estrogen regulated physiological functions of the female reproductive system. Several studies already aimed to investigate the impact of the AHR mediated xenobiotic response pathway on estrogen receptor (ER) signalling, whereas on contrary availability of data describing the effect of 17β-Estradiol (E2) on the AHR signalling cascade is rather limited. In this study we observed an inhibitory effect of E2 treatment on uterine Ahr, Arnt, Arnt2, Ahrr, Cyp1a1, Ugt1 and Nfe2l2 gene expression in ovariectomized Wistar rats, whereas Cyp1b1, Nqo1 and Gsta2 displayed an increased transcription. The usage of the ER selective agonists, 16α-LE(2) (ERα selective) and 8β-VE(2) (ERβ selective), enabled us to distinguish between ER subtype specific responses. On mRNA level the observed changes in gene expression were mainly mediated by ERα except for the expression of Nqo1. In most cases the activation of ERβ caused effects opposite to the ones observed following activation of ERα. Despite the significant changes in AHR mRNA levels immunohistochemical staining uterine tissue section did not reveal changes of the AHR protein level. Taken together our results validate, support and extend the hypothesis of uterine crosstalk between AHR and ER signalling pathways. Furthermore they give an insight into how the AHR and its related genes may participate in E2 dependent uterine physiological processes and provide another potential mechanism of action for xenoestrogens.

tert-Butylhydroquinone (TBHQ) is an antioxidant and preservative used in unsaturated vegetable oils and processed foods. However, when consumed in higher doses daily, it may pose a threat to public health by potentially increasing the risk of cancer, as it has an affinity with both the aryl hydrocarbon receptor (AhR) and the estrogen receptor alpha (ERα). This study aimed to examine the impact of substituting the 1,4-diol of TBHQ with 1,4-dithiol, referred to as TBDT, on the carcinogenic and antioxidant systems using computational methods. The binding affinity of TBHQ and TBDT to the two carcinogenic receptors, AhR and ERα, as well as to the antioxidant receptor Keap1 alone and in connection with Nrf2 (Nrf2-Keap1) was investigated through docking analysis. The results indicated a decrease in TBDT's binding strength to ERα and AhR when assessed using Molegro Virtual Docker (P-value: 0.0001 and 0.00001, respectively), AutoDock Vina (P-value: 0.0001 and 0.0001), and the online server Fast DRH (P-value: 0.0001 and 0.0001). However, TBDT's binding affinity to Keap1 was predicted to be significantly stronger than TBHQ's by both MVD and AutoDock Vina (P-value: 0.0001 and 0.04), while its binding to Nrf2-Keap1 assessed to be stronger only by MVD (P-value: 0.0001). These findings suggest that TBDT not only exhibits higher antioxidant activity as a better ligand for the antioxidant system but also shows lower affinity with the AhR and ERα receptors. Therefore, TBDT can be considered a safer compound than TBHQ.

The local interpretable model-agnostic explanation method was used to unveil substructures (toxic alerts) that cause endocrine disruption in chemical compounds using machine learning models. The random forest classifier was applied to build explainable models with the TOX21 data sets after data curation. Using these models applied to the EDC and EDKB-FDA data sets, the substructures that cause endocrine disruption in chemical compounds were unveiled, providing stable, more specific, and consistent explanations, which are essential for trust and acceptance of the findings, mainly due to the difficulty of finding relevant experimental evidence for different receptors (androgen, estrogen, aryl hydrocarbon, aromatase, and peroxisome proliferator-activated receptors). This approach is significant because of its contribution to the interpretability of explainable machine learning algorithms, particularly in the context of unveiling substructures associated with endocrine disruption in five targets (androgen receptor, estrogen receptor, aryl hydrocarbon receptors, aromatase receptors, and peroxisome proliferator-activated receptors), thereby advancing the relevant field of environmental toxicology, where a careful evaluation of the potential risks of exposure to new compounds is needed. The specific substructures thiophosphate, sulfamate, anilide, carbamate, sulfamide, and thiocyanate are presented as toxic alerts that cause endocrine disruption to better understand their potential risks and adverse effects on human health and the environment.