Isolation and Identification of Aryl Hydrocarbon Receptor Modulators

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Bioactive Constituents, Metabolites, and Functions

Isolation and Identification of Aryl Hydrocarbon Receptor Modulators in White Button Mushrooms (Agaricus bisporus) Yuan Tian, Wei Gui, Phillip B Smith, Imhoi Koo, Iain A. Murray, MT Cantorna, Gary H Perdew, and Andrew D. Patterson J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03212 • Publication Date (Web): 24 Jul 2019 Downloaded from pubs.acs.org on July 25, 2019

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Journal of Agricultural and Food Chemistry

Isolation and Identification of Aryl Hydrocarbon Receptor Modulators in White Button Mushrooms (Agaricus bisporus) †,‡,#

Yuan Tian

†#

§

†

†

Wei Gui , , Philip B. Smith , Imhoi Koo , Iain A. Murray , Margherita T. †

†

†,*

Cantorna , Gary H. Perdew , and Andrew D. Patterson

†Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802, United States ‡CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Centre for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, University of Chinese Academy of Sciences, Wuhan, 430071, P. R. China §Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802, United States

#These authors contributed equally to this work *To whom correspondence should be addressed. Email: [email protected]. Address: 322 Life Science Bldg, University Park, PA 16802, Phone: 814-867-4565

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ABSTRACT Natural aryl hydrocarbon (AHR) ligands have been identified in food and herbal medicines, and they may exhibit beneficial activity in humans. In this study, white button (WB) feeding significantly decreased AHR target gene expression in the small intestine of both conventional (CV) and germfree (GF) mice. HPLC fractionation and UHPLC-MS/MS combined with an AHRresponsive cell-based luciferase gene reporter assay were used to isolate and characterize benzothiazole (BT) derivatives and 6-methylisoquinoline (6-MIQ) as novel AHR modulators from WB mushrooms. The study showed dose-dependent changes of AHR transformation determined by cell-based luciferase gene reporter assay and transcription of CYP1A1 in human Caco-2 cell by BT derivatives and 6-MIQ. These findings suggested that WB mushroom contains new classes of natural AHR modulators and demonstrated that HPLC fractionation and UHPLC-MS/MS combined with cell-based luciferase gene reporter assay as a useful approach for isolation and characterization of the previously unidentifed AHR modulators from natural products.

KEYWORDS: white buttom mushroom, aryl hydrocarbon ligand, mass spectrometry


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INTRODUCTION The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates biological pathways including intestinal homeostasis, carcinogenesis, cell cycle, and immune function.1-3 In earlier studies, AHR was reported to be primarily involved in environmental toxicant metabolism.4-5 Recently, emerging evidence indicates that obesity may be associated with AHR signaling, which showed that the AHR antagonists α-naphthoflavone and CH-223191 significantly reduced obesity and ameliorated liver steatosis in mice fed a Western diet.6-7 Further, the beneficial effects of dietary broccoli, a rich source of natural AHR ligands,8 was found to support maintenance of intestinal homeostasis through AHR activation.9 Therefore, studies to further isolate and characterize the individual chemical components in foods that modulate AHR activity are needed. The identification of AHR ligands has extended the understanding of the bioactivity of AHR. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent ligand of AHR, has been demonstrated to induce biochemical and toxic effects that dramatically impact host metabolism.1011

Natural AHR agonists and antagonists have been identified, at varying levels, in food.12-14 The

natural AHR ligands from food and herbal medicines include flavonoids,15 indoles,8 and resveratrol,16 which are reported to play beneficial regulatory roles in the host. Therefore, identification and characterization of novel dietary AHR ligands could increase our understanding of the structural diversity and activity of natural AHR ligands. White button (WB) mushroom (Agaricus bisporis) represents 90% of the total mushroom intake according to the United States Department of Agriculture.17 We previously reported that WB feeding in mouse models modulated the microbiome and was associated with fewer inflammatory cells that reduced the incidence and severity of colitis in the gastrointestinal mucosa



following Citrobacter rodentium infection.18 Additionally, we demonstrated that WB feeding resulted in expanding a population of Prevotella that induced intestinal gluconeogenesis and improved glucose homeostasis.19 WB mushrooms contain various bioactive and antioxidant components including phenolic compounds,20 ergosterols,21 quaternary alkaloids,22 and indole alkaloids.23 However, to the best of our knowledge, no study has identified potential AHR ligands in WB mushrooms. Here, using cell-based luciferase gene reporter assays coupled with HPLC fractionation and UHPLC-MS/MS structural elucidation, we identified benzothiazole (BT) derivatives and 6methylisoquinoline (6-MIQ) as AHR modulators in WB mushroom extracts. This study suggests that WB mushrooms represent a previously uncharacterized dietary source with the capacity to influence AHR transcriptional activity. We demonstrated that HPLC fractionation and UHPLCMS/MS combined with cell-based luciferase gene reporter assays are useful in the isolation and characterization of new AHR ligands from natural products.


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MATERIALS and METHODS Chemicals. TCDD and CH-223191 were ordered from AccuStandard (New Haven, CT). The standards of potential AHR modulators including BT derivatives and 6-MIQ were obtained from Sigma-Aldrich Inc. (St. Louis, MO) and were of the highest purity available. Mice and Diets. Agaricus bisporous WB mushrooms were gifts from Giorgio Foods (Temple, PA). The mushrooms were freeze-dried and ground and sent to Teklad Diets (Madison, WI) to make WB diet at 1g/100g (1%). The WB and CTRL diets were irradiated to sterilize them and used for GF as well as CV mice. Mice were fed the weight equivalent to 1 human serving of whole WB mushrooms which equals 75-100 g fresh WB weight in a human diet.19 Animal experimental procedures were performed using protocols approved by the Pennsylvania State University Institutional Animal Care and Use Committee. Ten male C57BL/6 wild type mice were ordered from Jackson Laboratories (Bar Harbor, MN). Ten male GF mice were from the Pennsylvania State University Gnotobiotic Facility. Six weeks old mice were fed CTRL diets or WB diets for 2 wks. Cell Culture. HepG2 40/6 (human hepatoma cell line), Hepa 1.1 (mouse hepatoma cell line), and Caco-2 (human colon carcinoma cell line) were cultured in minimum essential medium eagle (Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA) and 1% penicillin (100 units/ml)/streptomycin (100 μg/ml) (Corning, Corning, NY). The cells were maintained at 37˚C in an atmosphere containing 5% CO2. RNA Isolation and Quantitative Real-time PCR. Caco-2 cells were cultured in 12-well plates and treated as indicated for 4 h. Total RNA was extracted from mice tissues (50 mg) and cultured cells (50,000 cells/well) using TRIzol reagent (Invitrogen, Carlsbad, CA). cDNA was synthesized from 1 μg RNA using qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD). qPCR



reactions were performed using SYBR green QPCR master mix with an ABI Prism 7900HT Fast real-time PCR sequence detection system (Applied Biosystems, Waltham, MA). The primers were listed in Supplementary Table S1 and data were normalized to Rpl13 (cell) or Gapdh (tissue) mRNA levels. Extraction and Fractionation of WB Mushroom Using HPLC. Dried mushroom powder (100 mg) was extracted twice using the Precellys tissue homogenizer (Bertin Technologies, Rockville, MD) with 3 mL of methanol (MeOH) or ethyl acetate. The combined extract was evaporated using nitrogen gas and reconstituted in 1 ml of DMSO (for cell luciferase reporter assay) or 50 μl of ethyl acetate (for HPLC fractionation). The fractionation of WB mushroom was performed on a Waters 2695 HPLC system (Waters, Milford, MA) with a Restek (Bellefonte, PA) HPLC C18 column (4.6x150 mm, 5 µm particle size) and a Viva C18 guard cartridge (10x4.0 mm, 5 µm particle size). The mobile phase solvent A was 2% (v/v) acetic acid and solvent B was 0.5% acetic acid in water and acetonitrile (50:50, v/v). The gradient program was followed previous study24 with minor modifications: 0–50 min, 10–55% B in A; 50–60 min, 55–100% B in A; 60–65 min, 100–10% B in A. The injection volume of mushroom extract was 10 μl. Simultaneous monitoring was performed at 254 nm at a flow rate of 1 ml/min. Fractions were collected 1.5 min by using Waters fraction collector ΙΙΙ (All waters, Milford, MA). Identification of AHR Modulators by UHPLC-Orbitrap Fusion-MS. The fractions with putative AHR modulators were evaporated under nitrogen gas and reconstituted in 50 µl of 3% methanol. The fractions and standards of potential AHR modulators that were obtained from Sigma-Aldrich Inc. (St. Louis, MO) were analyzed using a Vanquish UHPLC system connected to an Orbitrap Fusion Tribrid MS (ThermoFisher Scientific, Waltham, MA). Samples (5 µl) were separated by reverse phase HPLC using the Vanquish UPLC system (ThermoFisher) with a Waters


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(Milford, MA) BEH C18 column (100mm x 2.1mm, 1.7 µm particle size) maintained at 55˚C and a 20 minute aqueous acetonitrile gradient, at a flow rate of 250 µl/min. Solvent A was HPLC grade water with 0.1% formic acid and Solvent B was HPLC grade acetonitrile with 0.1% formic acid. The initial condition were 97% A and 3 % B, increasing to 45% B at 10 min, 75% B at 12 min where it was held at 75% B until 17.5 min before returning to the initial conditions. Potential AHR modulators were identified using Compound Discoverer (Thermo Fisher Scientific, Waltham, MA) and quantified using standard curves with concentrations ranging from 0.1 µM to 10 µM. The identification accuracies wer calculated between standards and mushroom extracts using Stein and Scott’s composite similarity score.25 Luciferase-based Reporter Assay. HepG2 40/6 and Hepa 1.1 cells were cultured in 24-well plates and were treated as indicated. After incubation, cell viability was checked using a microscope. Subsequently, cells were lysed with 100 μl of reporter lysis buffer [25mM Trisphosphate, pH 7.8, 2 mM dithiothreitol, 2 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100] and kept in -80˚C at least for 10 min. 80 μl of Luciferase Reporter Substrate (Promega, Madison, WI) and 20 μl of lysate from each well were combined and luciferase activity was determined using a GloMax® 20/20 luminometer (Promega, Madison, WI) and reported as relative light unit (RLU). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay. HepG2 40/6 cells were cultured in 96-well plates and treated as indicated for 5 h. Cell cytotoxicity was measured using the MTT assay kit (Abcam, Cambridge, MA) according to the manufacturer’s instructions. The absorbance was measured at 570 nm using a plater reader and the percent cytotoxicity was calculated as follows: 100 × (absorbance of reference group - absorbance of the experimental group)/(absorbance of reference group).



Statistics. Values are the means ± standard deviations (SD) or median and interquartile ranges. Graphical illustrations and statistical analysis were performed using GraphPad Prism (v 6.0, GraphPad). Statistical analyses were performed uing unpaired t-test analysis for the in vivo experiment and one-way analysis of variance followed by Dunnett’s multiple comparisions for the in vitro experiment.


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Results and Discussion AHR acts as a ligand-activated transcription factor and has been associated with obesity and metabolic disorders.1,

6

This is the first study to characterize and isolate potential AHR

modulators present in WB mushrooms. The workflow is shown in Figure 1. First, fractions of WB mushroom extract were collected via HPLC fractionation. Second, AHR activity of the concentrated fractions was measured by AHR-dependent cell-based luciferase gene reporter assay. Then, AHR modulator candidates from selected fractions were identified using UHPLC-MS/MS and their activity confirmed by an AHR-dependent cell-based luciferase gene reporter assay. WB Mushroom Exhibits Overall AHR Antagonistic Effects in Vitro and Vivo. Recently, natural AHR ligands were identified in food and herbal medicines including (-)Epigallocatechin gallate from green tea,12 indole-3-carbinol from broccoli,9 ginsenosides from ginseng,14 isoflavones from soybean,13 and chrysin from propolis.13 In order to analyze potential AHR activity of WB mushrooms, human HepG2 40/6 cells stably harboring an AHR responsive luciferase reporter construct were used to assess AHR transcriptional activity. As shown in Figure 2A-B, methanol (MeOH) or ethyl acetate extracts of WB mushrooms significantly inhibited TCDD-induced luciferase activity in a dose-dependent manner. Since the ethyl acetate extract showed stronger AHR activity and better reproducibility (Figure 2A-B), ethyl acetate was selected for further investigation in this study as the extraction solvent. To assess the cytotoxicity of the WB mushroom extract, the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reporter assay was used with varying dilutions of WB mushroom extract. Results from the MTT assay demonstrated that the mushroom extract did not result in extensive cytotoxicity (Figure S1). The physiologic impact of WB mushroom consumption associated with AHR activity were assessed in conventional (CV) and germfree (GF) mice. Two weeks of WB feeding in CV and GF mice significantly decreased mRNA expression for the AHR target genes in the small intestine



(Figure 2C-D), which suggests that the AHR activity of WB diets was independent of the microbiota. However, WB feeding had no effect on AHR target gene expression in the liver or colon from CV mice (Figure 2E), which suggested either that the effects of WB diets on AHR activity may be tissue specific or due to metabolism and clearance of AHR modulators within the intestinal tract. These data indicate that WB mushroom extract contains AHR modulators whose antagonistic activities can compete with agonistic activities both in vivo and in vitro. AHR responsiveness to dietary antagonists may be a significant contributor to WB mushroom-associated maintenance of intestinal homeostasis.19 Isolation and Identification of the AHR Modulators in WB Mushroom Extract. To identify and characterize the bioactive compounds, isolation and purification of the bioactive fractions from complex natural products is a critical step. HPLC has been widely applied for the isolation of ligands of enzymes, proteins, or receptors from natural product matrices,13 wastewater,26 cell,27 urine,28 and cecal contents.29 In this study, fractions of WB mushroom extract were collected after HPLC and the AHR transcriptional activity of the concentrated fractions were measured using the human HepG2 40/6 AHR-driven luciferase reporter cell line. Figure 3A shows the HPLC chromatogram of WB mushroom extract at UV absorbance 254 nm, which has been shown to be strongly correlated with the hydrophobic organic acid fraction.30 Figure 3B-C reveals the AHR agonist and antagonist activities of each fractions. Significant AHR activities were observed in 3 fractions (F1, F2, and F3) (Figure 3B), and inhibition of TCDD-induced AHR activity by 1 fraction (F4) was observed (Figure 3C). These data confirmed that WB mushroom extract contains natural AHR modulators with agonist and antagonist-like activities. Fractions (F1, F2, F3, and F4) containing AHR modulator candidates were analyzed by UHPLC-Oribtrap Fusion Lumos with MSn capabilities. In total, 5 benzothiazole (BT) derivatives


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including

BT,

2-aminobenzothiazole

(2-ABT),

2-mercaptobenzothiazole

(2-MBT),

2-

hydroxybenzothiazole (2-HBT), and 2,2′-dithiobis(benzothiazole) (2,2′-DBT), as well as 6methylisoquinoline (6-MIQ) in WB mushroom were identified (Figure 4A). The retention time and MS/MS spectra (positive ion mode) from authentic standards and WB mushroom extract are compared in Figure 4B-C and Figure S2. The chemical formulas, retention time (RT), ionization products [M+H]+, mass errors, and the contents of AHR modulators in WB mushroom extract obtained via UHPLC-MS/MS are summarized in Table 1. Compound identification between standards and WB mushroom extract was measured using Stein and Scott’s Composite Similarity as shown in Table 2. AHR Activity of Potential Modulators. BT constituents of many bioactive heterocyclic compounds that were first isolated from American cranberries.31 BT derivatives have gained increasing attention because of their diverse biological activities including anti-tumor,32-33 antimicrobial,34 anti-inflammatory,35 anti-diabetic,36 anti-tubercular,37 anti-convulsant,38 and use as analgesics.39 Recently, emerging research has focused on the utility of BT derivatives to improve anti-cancer drug activities.32, 40-41 A previous study isolated BT derivatives including BT, 2-MBT, and 2-HBT from rubber tires and identified 2-MBT as an AHR agonist.42 Quinoline is a heterocyclic aromatic organic compound, derivatives of which have been identified in natural products

43

and body fluids.44 Similar to BT, quinoline and its derivatives also have diverse

biological properties 45-47 and play an important role in developing anti-tumor drugs perhaps due to its interaction with enzymes like gyrase, topoisomerase II and other kinases.48 Recent reports indicate that 2,8-dihydroxyquionline, an AHR ligand, might have anti-inflammatory effects due to its potential to induce AHR activity in the intestine.49



To confirm the AHR activities of BT and quinoline derivatives isolated from WB mushroom, AHR-responsive cell-based luciferase gene reporter assays were performed using both human (HepG2 40/6) and mouse (Hepa 1.1) cells treated with increasing doses of BT and quinoline derivatives as well as TCDD as a control. BT derivatives with the exception of BT were able to induce AHR-mediated transcription significantly at a dose of 10 μM in both human (Figure 5A) and mouse (Figure S3A) cells but failed to show antagonist activity (Figure S4A), which suggested that BT derivatives may be activators of both human and mouse AHR. These data are consistent with the observation of induction of luciferase activity by 10 μM 2-MBT extracted from rubber tires.49 BT derivatives-induced luciferase activities were significantly inhibited by CH-223191, a potent AHR antagonist (Figure 5B), which further supported that BT derivatives have AHR agonistic activity. AHR is a ligand-activated transcription factor, most notably regulating enzymes involved with phase I metabolism such as modulating transcription of genes including CYP1A1.50 Here, BT derivatives with the exception of BT induced AHR activity in HepG2 40/6 cell and CYP1A1 in Caco-2 cell in a dose-dependent manner (Figure 5C-D). An MTT assay was conducted to assess the cytotoxicity of BT derivatives on HepG2 40/6 cells and demonstrate that the toxicity of BT derivatives were only observed at doses greater than 100 M (Figure S5A). These results indicated that BT derivatives including 2-ABT, 2-MBT, 2-HBT, and 2,2′-DBT are activators of both human and mouse AHR. The reporter assay was also conducted to determine the AHR antagonist activity of 6-MIQ. In both human (Figure 6A) and mouse (Figure S4B) cells, 6-MIQ was able to significantly inhibit TCDD-induced transcriptional activity of the AHR at a dose of 10 μM but failed to show agonist activity (Figure S4B). Moreover, a dose response study also showed dose-dependent inhibition of


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TCDD-induced AHR transformation in HepG2 40/6 cell and transcription of CYP1A1 in Caco-2 cell by 6-MIQ (Figure 6B-C). The MTT assay showed significant cytotoxicity of 6-MIQ at doses greater than 100 μM (Figure S5B). These data confirmed 6-MIQ as a potential human and mouse AHR inhibitor. In conclusion, this study used a combination of AHR-responsive cell-based luciferase gene reporter assays coupled with HPLC fractionation and UHPLC-MS/MS to isolate and characterize BT derivatives including 2-ABT, 2-MBT, 2-HBT, and 2,2′-DBT and 6-MIQ as natural AHR modulators present in WB mushroom. These data increase our understanding of the diversity of potential AHR modulators in food and suggest that WB mushroom might represent a dietary source of natural AHR modulators. Further work is needed to determine the physiological impacts of BT and quinoline derivatives as potential AHR modulators in vivo. In addition, the source of overall antagonistic activity observed in WB mushrooms remains to be identified.



ASSOCIATED CONTENT Supporting Information mRNA gene-targeted primers (Table S1), MTT assay (Figure S1 and S5), UHPLC/MS-MS chromatograms (Figure S2), Hepa 1.1 reporter cells (Figure S3), HepG2 40/6 reporter cells (Figure S4)


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AUTHOR INFORMATION Corresponding Author *Tel: +18148674565. E-mail: [email protected] ORCID Yuan Tian: 0000-0001-6174-3359 Andrew D. Patterson: 0000-0003-2073-0070 Author Contributions #

Yuan Tian and Wei Gui contributed equally to this work.

Notes The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported in part by the National Institutes of Health grants ES028288 (ADP), ES026684 (ADP), ES028244 (GHP), the Pennsylvania Department of Health using Tobacco CURE funds. This work was also supported by the USDA National Institute of Food and Federal Appropriations under Project PEN04607 and Accession number 1009993 (ADP, MTC, GHP).


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30. Hansen, A. M.; Kraus, T. E. C.; Pellerin, B. A.; Fleck, J. A.; Downing, B. D.; Bergamaschi, B. A., Optical properties of dissolved organic matter (DOM): Effects of biological and photolytic degradation. Limnol Oceanogr 2016, 61 (3), 1015-1032. 31. K, A.; E, v. S., The aroma of cranberries. II. Vaccinium macrocarpon Ait. Acta Chem. Scand. 1967, 21, 2076-2082. 32. Singh, M.; Singh, S. K., Benzothiazoles: How Relevant in Cancer Drug Design Strategy? Anti-Cancer Agents Med. Chem. 2014, 14 (1), 127-146. 33. Li, H. S.; Wang, X. R.; Duan, G. Y.; Xia, C. C.; Xiao, Y. L.; Li, F. R.; Ge, Y. Q.; You, G. R.; Han, J. F.; Fu, X. P.; Tan, S. H.; Wang, R. W., Synthesis, Antitumor Activity and Preliminary Structure-activity Relationship of 2-Aminothiazole Derivatives. Chem. Res. Chin. Univ. 2016, 32 (6), 929-937. 34. Catalano, A.; Carocci, A.; Defrenza, I.; Muraglia, M.; Carrieri, A.; Van Bambeke, F.; Rosato, A.; Corbo, F.; Franchini, C., 2-Aminobenzothiazole derivatives: Search for new antifungal agents. Eur. J. Med. Chem. 2013, 64, 357-364. 35. Gurupadayya, B. M.; Gopal, M.; Padmashali, B.; Valdya, V. P., Synthesis and biological activities of fluoro benzothiazoles. Indian J. Heterocy. Chem. 2005, 15 (2), 169-172. 36. Meltzer-Mats, E.; Babai-Shani, G.; Pasternak, L.; Uritsky, N.; Getter, T.; Viskind, O.; Eckel, J.; Cerasi, E.; Senderowitz, H.; Sasson, S.; Gruzman, A., Synthesis and Mechanism of Hypoglycemic Activity of Benzothiazole Derivatives. J. Med. Chem. 2013, 56 (13), 5335-5350. 37. Telvekar, V. N.; Bairwa, V. K.; Satardekar, K.; Bellubi, A., Novel 2-(2-(4aryloxybenzylidene) hydrazinyl)benzothiazole derivatives as anti-tubercular agents. Bioorganic Med. Chem. Lett. 2012, 22 (1), 649-652. 38. Siddiqui, N.; Ahsan, W., Benzothiazole Incorporated Barbituric Acid Derivatives: Synthesis and Anticonvulsant Screening. Arch. Pharm. 2009, 342 (8), 462-468. 39. Palagiano, F.; Arenare, L.; DeCaprariis, P.; Grandolini, G.; Ambrogi, V.; Perioli, L.; Filippelli, W.; Falcone, G.; Rossi, F., Synthesis and SAR study of imidazo 2,1-b benzothiazole acids and some related compounds with anti-inflammatory and analgesic activities. Farmaco 1996, 51 (7), 483-491. 40. Noolvi, M. N.; Patel, H. M.; Kaur, M., Benzothiazoles: Search for anticancer agents. Eur. J. Med. Chem. 2012, 54, 447-462. 41. Kok, S. H. L.; Gambari, R.; Chui, C. H.; Yuen, C. W. M.; Lin, E.; Wong, R. S. M.; Lau, F. Y.; Cheng, G. Y. M.; Lam, W. S.; Chan, S. H.; Lam, K. H.; Cheng, C. H.; Lai, P. B. S.; Yu, M. W. Y.; Cheung, F.; Tang, J. C. O.; Chan, A. S. C., Synthesis and anti-cancer activity of benzothiazole containing phthalimide on human carcinoma cell lines. Bioorganic Med. Chem. 2008, 16 (7), 3626-3631. 42. He, G. C.; Zhao, B.; Denison, M. S., Identification of benzothiazole derivatives and polycyclic aromatic hydrocarbons as aryl hydrocarbon receptor agonists present in tire extracts. Environ. Toxicol. Chem. 2011, 30 (8), 1915-1925. 43. Michael, J. P., Quinoline, quinazoline and acridone alkaloids. Nat. Prod. Rep. 2008, 25 (1), 166-187. 44. Li, F.; Pang, X. Y.; Krausz, K. W.; Jiang, C. T.; Chen, C.; Cook, J. A.; Krishna, M. C.; Mitchell, J. B.; Gonzalez, F. J.; Patterson, A. D., Stable Isotope- and Mass Spectrometry-based Metabolomics as Tools in Drug Metabolism: A Study Expanding Tempol Pharmacology. J. Proteome Res. 2013, 12 (3), 1369-1376.



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Table 1. AHR modulators identified in WB mushroom extract by UHPLC-MS/MS. Compound

Formula

RT

Theoretical m/z

Measured m/z

Mass error

Fraction

Contents in WB

(min)

of [M+H]+ ion

of [M+H]+ ion

(ppm)

No.

(µmol/mg dry weight)

Benzothiazole (BT)

C7H5NS

8.00

136.0220

136.0217

-2.21

F1,F2

70.5

2-Aminobenzothiazole (2-ABT)

C7H6N2S

3.81

151.0330

151.0325

-3.31

F1,F2

6.8

2-Mercaptobenzothiazole (2-

C7H5NS2

8.43

167.9942

167.9936

-3.57

F1,F2

4.6

2-Hydroxybenzothiazole (2-HBT)

C7H5NOS

7.30

152.0170

152.0166

-2.63

F1,F2

26.0

2,2′-Dithiobis(benzothiazole)

C14H8N2S4

14.98

332.9649

332.9642

-2.10

F3

49.8

C10H9N

3.98

144.0813

144.0810

-2.08

F4

2.8

MBT)

(2,2′-DBT) 6-methylisoquinoline (6-MIQ)



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Table 2. Similarities scores between standards and WB mushroom extract. Similarity score

BT

2-ABT

2-HBT

2-MBT

2,2'-DBT

6-MIQ

dot product

960

766

867

962

925

797

rev-dot product

973

974

963

967

955

869


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Figure 1. The flow diagram of isolation and identification of AHR modulators in WB mushroom.

Figure 2. (A-B) AHR antagonist activity of WB mushroom extraction. HepG2 40/6 reporter cells were treated with 5 nM TCDD and different concentrations of WB mushroom extraction for 5 h. Values are the mean ± S.D. of n = 3 per group. ***p < 0.001, ****p < 0.0001 compare to TCDD. (C-D) qPCR analysis of mRNA levels of AHR targeted genes in the duodenum and ileum from CV (C) and GF (D) mice after 2 weeks CTRL or WB feeding. (E) mRNA levels of CYP1A1 and CYP1A2 in the colon and liver from CV mice after 2 weeks CTRL or WB feeding. Values are the median and interquartile ranges of n = 5 mice per group. *p < 0.05, **p < 0.01 compare to CTRL. MeOH, methanol.

Figure 3. (A) HPLC chromatogram (UV absorbance at 254 nm versus retention time) of WB mushroom extraction. (B-C) HPLC fractionation of putative AHR modulators from WB mushroom extraction. HepG2 40/6 reporter cells were treated with HPLC fractions alone (B) or with 5 nM TCDD (C) for 5 h.

Figure 4. (A) Chemical structures of identified AHR modulators in WB mushroom. (B-C) UHPLC/MS-MS chromatograms of standards 2-ABT (B) and 6-MIQ (C) and from WB mushroom extract.

Figure 5. (A-B) AHR agonistic activities of 10 µM BT derivatives alone or with 1 µM CH-223191 in HepG2 40/6 reporter cells. (C) Dose-response curves of AHR agonistic activity of BT derivatives in HepG2 40/6 reporter cells. (D) Dose-dependent induction of CYP1A1 expression of



BT derivatives or 10 nM TCDD mediated by the human AHR in Caco-2 cells. Values are the mean ± S.D. of n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Figure 6. (A-B) AHR antagonist activity of 6-MIQ. HepG2 40/6 reporter cells were treated with 5 nM TCDD and 10 µM or different concentrations of 6-MIQ for 5 h. (C) Dose-dependent induction of CYP1A1 expression of identified compounds or 10 nM TCDD mediated by the human AHR in Caco-2 cells. Values are the mean ± S.D. of n = 3 per group. **p < 0.01, ****p < 0.0001


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TOC



Figure 1. The flow diagram of isolation and identification of AHR modulators in WB mushroom. 269x147mm (300 x 300 DPI)


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Figure 2. (A-B) AHR antagonist activity of WB mushroom extraction. HepG2 40/6 reporter cells were treated with 5 nM TCDD and different concentrations of WB mushroom extraction for 5 h. Values are the mean ± S.D. of n = 3 per group. ***p < 0.001, ****p < 0.0001 compare to TCDD. (C-D) qPCR analysis of mRNA levels of AHR targeted genes in the duodenum and ileum from CV (C) and GF (D) mice after 2 weeks CTRL or WB feeding. (E) mRNA levels of CYP1A1 and CYP1A2 in the colon and liver from CV mice after 2 weeks CTRL or WB feeding. Values are the median and interquartile ranges of n = 5 mice per group. *p < 0.05, **p < 0.01 compare to CTRL. MeOH, methanol.



Figure 3. (A) HPLC chromatogram (UV absorbance at 254 nm versus retention time) of WB mushroom extraction. (B-C) HPLC fractionation of putative AHR modulators from WB mushroom extraction. HepG2 40/6 reporter cells were treated with HPLC fractions alone (B) or with 5 nM TCDD (C) for 5 h. 280x207mm (300 x 300 DPI)


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Figure 4. (A) Chemical structures of identified AHR modulators in WB mushroom. (B-C) UHPLC/MS-MS chromatograms of standards 2-ABT (B) and 6-MIQ (C) and from WB mushroom extract.



Figure 5. (A-B) AHR agonistic activities of 10 µM BT derivatives alone or with 1 µM CH-223191 in HepG2 40/6 reporter cells. (C) Dose-response curves of AHR agonistic activity of BT derivatives in HepG2 40/6 reporter cells. (D) Dose-dependent induction of CYP1A1 expression of BT derivatives or 10 nM TCDD mediated by the human AHR in Caco-2 cells. Values are the mean ± S.D. of n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 325x186mm (300 x 300 DPI)


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Figure 6. (A-B) AHR antagonist activity of 6-MIQ. HepG2 40/6 reporter cells were treated with 5 nM TCDD and 10 µM or different concentrations of 6-MIQ for 5 h. (C) Dose-dependent induction of CYP1A1 expression of identified compounds or 10 nM TCDD mediated by the human AHR in Caco-2 cells. Values are the mean ± S.D. of n = 3 per group. **p < 0.01, ****p < 0.0001 325x117mm (300 x 300 DPI)


Isolation and Identification of Aryl Hydrocarbon Receptor Modulators

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