Isolation and Identification of Aryl Hydrocarbon Receptor Modulators in White Button Mushrooms (Agaricus bisporus)
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 AHR-responsive 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.
INTRODUCTION<!>Chemicals.<!>Mice and Diets.<!>Cell Culture.<!>RNA Isolation and Quantitative Real-time PCR.<!>Extraction and Fractionation of WB Mushroom Using HPLC.<!>Identification of AHR Modulators by UHPLC-Orbitrap Fusion-MS.<!>Luciferase-based Reporter Assay.<!>3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay.<!>Statistics.<!>Results and Discussion<!>WB Mushroom Exhibits Overall AHR Antagonistic Effects in Vitro and Vivo.<!>Isolation and Identification of the AHR Modulators in WB Mushroom Extract.<!>AHR Activity of Potential Modulators.
<p>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.</p><p>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.10–11 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.</p><p>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.</p><p>Here, using cell-based luciferase gene reporter assays coupled with HPLC fractionation and UHPLC-MS/MS structural elucidation, we identified benzothiazole (BT) derivatives and 6-methylisoquinoline (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 UHPLC-MS/MS combined with cell-based luciferase gene reporter assays are useful in the isolation and characterization of new AHR ligands from natural products.</p><!><p>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.</p><!><p>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.</p><!><p>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.</p><!><p>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.</p><!><p>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 (4.6×150 mm, 5 μm particle size) and a Viva C18 guard cartridge (10×4.0 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 III (All waters, Milford, MA).</p><!><p>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 (Milford, MA) BEH C18 column (100mm × 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</p><!><p>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 Tris-phosphate, 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).</p><!><p>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).</p><!><p>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.</p><!><p>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.</p><!><p>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).</p><p>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</p><!><p>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.</p><p>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 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 6-methylisoquinoline (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.</p><!><p>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 anti-microbial,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 products43 and body fluids.44 Similar to BT, quinoline and its derivatives also have diverse biological properties45–47 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</p><p>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.</p><p>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 μM (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.</p><p>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 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.</p><p>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.</p>