The human aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is a pivotal regulator of human physiology and pathophysiology. Allosteric inhibition of AhR was previously thought to be untenable. Here, we identify carvones as noncompetitive, insurmountable antagonists of AhR and characterize the structural and functional consequences of their binding. Carvones do not displace radiolabeled ligands from binding to AhR but instead bind allosterically within the bHLH/PAS-A region of AhR. Carvones do not influence the translocation of ligand-activated AhR into the nucleus but inhibit the heterodimerization of AhR with its canonical partner ARNT and subsequent binding of AhR to the promoter of CYP1A1. As a proof of concept, we demonstrate physiologically relevant Ahr-antagonism by carvones in vivo in female mice. These substances establish the molecular basis for selective targeting of AhR regardless of the type of ligand(s) present and provide opportunities for the treatment of disease processes modified by AhR.

• MeSH
• Cytochrome P-450 CYP1A1 genetics   MeSH
• Skin * metabolism   radiation effects   MeSH
• Ligands MeSH
• Mice MeSH
• Promoter Regions, Genetic MeSH
• Aryl Hydrocarbon Receptor Nuclear Translocator * genetics   metabolism   MeSH
• Receptors, Aryl Hydrocarbon * genetics   metabolism   MeSH
• Ultraviolet Rays adverse effects   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytochrome P-450 CYP1A1 MeSH
• Ligands MeSH
• Aryl Hydrocarbon Receptor Nuclear Translocator * MeSH
• Receptors, Aryl Hydrocarbon * MeSH

We examined the effects of gut microbial catabolites of tryptophan on the aryl hydrocarbon receptor (AhR). Using a reporter gene assay, we show that all studied catabolites are low-potency agonists of human AhR. The efficacy of catabolites differed substantially, comprising agonists with no or low (i3-propionate, i3-acetate, i3-lactate, i3-aldehyde), medium (i3-ethanol, i3-acrylate, skatole, tryptamine), and high (indole, i3-acetamide, i3-pyruvate) efficacies. We displayed ligand-selective antagonist activities by i3-pyruvate, i3-aldehyde, indole, skatole, and tryptamine. Ligand binding assay identified low affinity (skatole, i3-pyruvate, and i3-acetamide) and very low affinity (i3-acrylate, i3-ethanol, indole) ligands of the murine AhR. Indole, skatole, tryptamine, i3-pyruvate, i3-acrylate, and i3-acetamide induced CYP1A1 mRNA in intestinal LS180 and HT-29 cells, but not in the AhR-knockout HT-29 variant. We observed a similar CYP1A1 induction pattern in primary human hepatocytes. The most AhR-active catabolites (indole, skatole, tryptamine, i3-pyruvate, i3-acrylate, i3-acetamide) elicited nuclear translocation of the AhR, followed by a formation of AhR-ARNT heterodimer and enhanced binding of the AhR to the CYP1A1 gene promoter. Collectively, we comprehensively characterized the interactions of gut microbial tryptophan catabolites with the AhR, which may expand the current understanding of their potential roles in intestinal health and disease.

• Keywords
• aryl hydrocarbon receptor, indoles, microbiome, tryptophan,
• MeSH
• Cytochrome P-450 CYP1A1 genetics   MeSH
• Gene Expression MeSH
• Indoles MeSH
• Humans MeSH
• Ligands MeSH
• Metabolic Networks and Pathways MeSH
• Protein Multimerization MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Promoter Regions, Genetic MeSH
• Receptors, Aryl Hydrocarbon agonists   metabolism   MeSH
• Genes, Reporter MeSH
• Gastrointestinal Microbiome * drug effects   MeSH
• Basic Helix-Loop-Helix Transcription Factors agonists   metabolism   MeSH
• Tryptophan metabolism   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• AHR protein, human MeSH Browser
• CYP1A1 protein, human MeSH Browser
• Cytochrome P-450 CYP1A1 MeSH
• indole MeSH Browser
• Indoles MeSH
• Ligands MeSH
• Receptors, Aryl Hydrocarbon MeSH
• Basic Helix-Loop-Helix Transcription Factors MeSH
• Tryptophan MeSH

Mono-methylindoles (MMI) were described as agonists and/or antagonists of the human aryl hydrocarbon receptor (AhR). Here, we investigated the effects of MMI on AhR-CYP1A pathway in human hepatocytes and HepaRG cells derived from human progenitor hepatic cells. All MMI, except of 2-methylindole, strongly induced CYP1A1 and CYP1A2 mRNAs in HepaRG cells. Induction of CYP1A genes was absent in AhR-knock-out HepaRG cells. Consistently, CYP1A1 and CYP1A2 mRNAs and proteins were induced by all MMIs (except 2-methylindole), in human hepatocytes. The enzyme activity of CYP1A1 was inhibited by MMIs in human hepatocytes and LS180 colon cancer cells in a concentration-dependent manner (IC50 values from 1.2 μM to 23.8 μM and from 3.4 μM to 11.4 μM, respectively). Inhibition of CYP1A1 activity by MMI in human liver microsomes was much weaker as compared to that in intact cells. Incubation of parental MMI with human hepatocytes either diminished (4-methylindole, 6-methylindole) or enhanced (7-methylindole) their agonist effects on AhR in AZ-AHR reporter cells. In conclusion, overall effects of MMI on AhR-CYP1A pathway in human cells comprise the induction of CYP1A genes through AhR, the inhibition of CYP1A catalytic activity and possibly the metabolic transformation causing loss or gain of AhR agonist activity of parental compounds.

• Keywords
• Aryl hydrocarbon receptor, Entero-hepatic axis, Methylindoles, Microbial catabolites, Tryptophan,
• MeSH
• Cytochrome P-450 CYP1A1 antagonists & inhibitors   biosynthesis   genetics   MeSH
• Enzyme Induction MeSH
• Hepatocytes drug effects   enzymology   MeSH
• Indoles pharmacology   MeSH
• Cytochrome P-450 Enzyme Inducers pharmacology   MeSH
• Cytochrome P-450 Enzyme Inhibitors pharmacology   MeSH
• Microsomes, Liver drug effects   enzymology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Cell Line, Tumor MeSH
• Colonic Neoplasms enzymology   MeSH
• Receptors, Aryl Hydrocarbon agonists   genetics   metabolism   MeSH
• Aged MeSH
• Basic Helix-Loop-Helix Transcription Factors agonists   genetics   metabolism   MeSH
• Dose-Response Relationship, Drug MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• AHR protein, human MeSH Browser
• CYP1A1 protein, human MeSH Browser
• Cytochrome P-450 CYP1A1 MeSH
• Indoles MeSH
• Cytochrome P-450 Enzyme Inducers MeSH
• Cytochrome P-450 Enzyme Inhibitors MeSH
• Receptors, Aryl Hydrocarbon MeSH
• Basic Helix-Loop-Helix Transcription Factors MeSH

Novel methylindoles were identified as endobiotic and xenobiotic ligands of the human aryl hydrocarbon receptor (AhR). We examined the effects of 22 methylated and methoxylated indoles on the transcriptional activity of AhRs. Employing reporter gene assays in AZ-AHR transgenic cells, we determined full agonist, partial agonist, or antagonist activities of tested compounds, having substantially variable EC50, IC50, and relative efficacies. The most effective agonists (EMAX relative to 5 nM dioxin) of the AhR were 4-Me-indole (134%), 6-Me-indole (91%), and 7-MeO-indole (80%), respectively. The most effective antagonists of the AhR included 3-Me-indole (IC50; 19 μM), 2,3-diMe-indole (IC50; 11 μM), and 2,3,7-triMe-indole (IC50; 12 μM). Reverse transcription polymerase chain reaction analyses of CYP1A1 mRNA in LS180 cells confirmed the data from gene reporter assays. The compound leads, 4-Me-indole and 7-MeO-indole, induced substantial nuclear translocation of the AhR and enriched binding of the AhR to the CYP1A1 promoter, as observed using fluorescent immunohistochemistry and chromatin immunoprecipitation assays, respectively. Molecular modeling and docking studies suggest the agonists and antagonists likely share the same binding pocket but have unique binding modes that code for their affinity. Binding pocket analysis further revealed that 4-methylindole and 7-methoxyindole can simultaneously bind to the pocket and produce synergistic interactions. Together, these data show a dependence on subtle and specific chemical indole structures as AhR modulators and furthermore underscore the importance of complete evaluation of indole compounds as nuclear receptor ligands.

• MeSH
• Hep G2 Cells MeSH
• Cytochrome P-450 CYP1A1 metabolism   MeSH
• Indoles pharmacology   MeSH
• Humans MeSH
• Ligands MeSH
• RNA, Messenger metabolism   MeSH
• Cell Line, Tumor MeSH
• Promoter Regions, Genetic drug effects   MeSH
• Receptors, Aryl Hydrocarbon agonists   antagonists & inhibitors   MeSH
• Genes, Reporter drug effects   MeSH
• Basic Helix-Loop-Helix Transcription Factors agonists   antagonists & inhibitors   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• AHR protein, human MeSH Browser
• Cytochrome P-450 CYP1A1 MeSH
• indole MeSH Browser
• Indoles MeSH
• Ligands MeSH
• RNA, Messenger MeSH
• Receptors, Aryl Hydrocarbon MeSH
• Basic Helix-Loop-Helix Transcription Factors MeSH

Khellin and visnagin are two furanochromones that can be frequently found in ethnomedical formulations in Asia and the Middle East. Both compounds possess anti-inflammatory and analgesic properties, therefore modern medicine uses these compounds or structurally related derivatives for treatment of vitiligo, bronchial asthma and renal colics. Despite their frequent usage, the potential toxic properties of visnagin and khellin are not well characterized up-to-now. Many natural compounds modulate the expression and activity of cytochrome P450 1A1 (CYP1A1), which is well-known to bioactivate pro-carcinogens. The expression of this enzyme is controlled by the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor and regulator of drug metabolism. Here, we investigated the influence of both furanochromones on AHR signaling in human HepG2 hepatocarcinoma cells and primary human hepatocytes. Both compounds transactivated xenobiotic response element (XRE)-driven reporter gene activity in a dose-dependent manner and induced CYP1A1 transcription in HepG2 cells and primary hepatocytes. The latter was abolished in presence of a specific AHR antagonist. CYP1A enzyme activity assays done in HepG2 cells and primary hepatocytes revealed an inhibition of enzyme activity by both furanochromones, which may become relevant regarding the metabolism of xenobiotics and co-administered therapeutic drugs. The observed induction of several other members of the AHR gene battery, whose gene products are involved in regulation of cell growth, differentiation and migration, indicates that a further toxicological characterization of visnagin and khelllin is urgently required in order to minimize potential drug-drug interactions and other toxic side-effects that may occur during therapeutic usage of these furanochromones.

• MeSH
• Enzyme Activation drug effects   MeSH
• Aryl Hydrocarbon Hydroxylases metabolism   MeSH
• Cell Line MeSH
• Cytochrome P-450 CYP1A1 genetics   metabolism   MeSH
• Gene Expression MeSH
• Hepatocytes drug effects   metabolism   MeSH
• Khellin pharmacology   MeSH
• Humans MeSH
• Receptors, Aryl Hydrocarbon metabolism   MeSH
• Gene Expression Regulation drug effects   MeSH
• Genes, Reporter MeSH
• Signal Transduction drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Aryl Hydrocarbon Hydroxylases MeSH
• Cytochrome P-450 CYP1A1 MeSH
• Khellin MeSH
• Receptors, Aryl Hydrocarbon MeSH
• visnagin MeSH Browser

We examined the effects of anthocyanidins (cyanidin, delphinidin, malvidin, peonidin, petunidin, pelargonidin) on the aryl hydrocarbon receptor (AhR)-CYP1A1 signaling pathway in human hepatocytes, hepatic HepG2 and intestinal LS174T cancer cells. AhR-dependent reporter gene expression in transfected HepG2 cells was increased by pelargonidin in a concentration-dependent manner at 24h. Similarly, pelargonidin induced the expression of CYP1A1 mRNA up to 5-fold in HepG2 and LS174T cells relative to the induction by 5 nM 2,3,7,8-tetrachlorodibenzodioxin (TCDD), the most potent activator of AhR. CYP1A1 and CYP1A2 mRNAs were also increased by pelargonidin in three primary human hepatocytes cultures (approximately 5% of TCDD potency) and the increase in CYP1A1 protein in HepG2 and LS174T cells was comparable to the increase in catalytic activity of CYP1A1 enzyme. Ligand binding analysis demonstrated that pelargonidin was a weak ligand of AhR. Enzyme kinetic analyses using human liver microsomes revealed inhibition of CYP1A1 activity by delphinidin (IC50 78 μM) and pelargonidin (IC50 33 μM). Overall, although most anthocyanidins had no effects on AhR-CYP1A1 signaling, pelargonidin can bind to and activate the AhR and AhR-dependent gene expression, and pelargonidin and delphinidin inhibit the CYP1A1 catalytic activity.

• MeSH
• Transcriptional Activation drug effects   MeSH
• Anthocyanins pharmacology   MeSH
• Hep G2 Cells MeSH
• Cytochrome P-450 CYP1A1 biosynthesis   MeSH
• Enzyme Induction MeSH
• Hepatocytes drug effects   enzymology   MeSH
• Microsomes, Liver enzymology   MeSH
• Kinetics MeSH
• Humans MeSH
• Ligands MeSH
• RNA, Messenger biosynthesis   MeSH
• Liver Neoplasms enzymology   MeSH
• Polychlorinated Dibenzodioxins pharmacology   MeSH
• Primary Cell Culture MeSH
• Promoter Regions, Genetic drug effects   MeSH
• Receptors, Aryl Hydrocarbon drug effects   metabolism   MeSH
• Signal Transduction drug effects   MeSH
• Intestinal Neoplasms enzymology   MeSH
• Transfection MeSH
• Basic Helix-Loop-Helix Transcription Factors drug effects   metabolism   MeSH
• Dose-Response Relationship, Drug MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• AHR protein, human MeSH Browser
• Anthocyanins MeSH
• CYP1A1 protein, human MeSH Browser
• Cytochrome P-450 CYP1A1 MeSH
• Ligands MeSH
• RNA, Messenger MeSH
• pelargonidin MeSH Browser
• Polychlorinated Dibenzodioxins MeSH
• Receptors, Aryl Hydrocarbon MeSH
• Basic Helix-Loop-Helix Transcription Factors MeSH