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      Skatole (3-Methylindole) Is a Partial Aryl Hydrocarbon Receptor Agonist and Induces CYP1A1/2 and CYP1B1 Expression in Primary Human Hepatocytes

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          Abstract

          Skatole (3-methylindole) is a product of bacterial fermentation of tryptophan in the intestine. A significant amount of skatole can also be inhaled during cigarette smoking. Skatole is a pulmonary toxin that induces the expression of aryl hydrocarbon receptor (AhR) regulated genes, such as cytochrome P450 1A1 (CYP1A1), in human bronchial cells. The liver has a high metabolic capacity for skatole and is the first organ encountered by the absorbed skatole; however, the effect of skatole in the liver is unknown. Therefore, we investigated the impact of skatole on hepatic AhR activity and AhR-regulated gene expression. Using reporter gene assays, we showed that skatole activates AhR and that this is accompanied by an increase of CYP1A1, CYP1A2 and CYP1B1 expression in HepG2-C3 and primary human hepatocytes. Specific AhR antagonists and siRNA-mediated AhR silencing demonstrated that skatole-induced CYP1A1 expression is dependent on AhR activation. The effect of skatole was reduced by blocking intrinsic cytochrome P450 activity and indole-3-carbinole, a known skatole metabolite, was a more potent inducer than skatole. Finally, skatole could reduce TCDD-induced CYP1A1 expression, suggesting that skatole is a partial AhR agonist. In conclusion, our findings suggest that skatole and its metabolites affect liver homeostasis by modulating the AhR pathway.

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          Cytochrome p450 and chemical toxicology.

          The field of cytochrome P450 (P450) research has developed considerably over the past 20 years, and many important papers on the roles of P450s in chemical toxicology have appeared in Chemical Research in Toxicology. Today, our basic understanding of many of the human P450s is relatively well-established, in terms of the details of the individual genes, sequences, and basic catalytic mechanisms. Crystal structures of several of the major human P450s are now in hand. The animal P450s are still important in the context of metabolism and safety testing. Many well-defined examples exist for roles of P450s in decreasing the adverse effects of drugs through biotransformation, and an equally interesting field of investigation is the bioactivation of chemicals, including drugs. Unresolved problems include the characterization of the minor "orphan" P450s, ligand cooperativity and kinetic complexity of several P450s, the prediction of metabolism, the overall contribution of bioactivation to drug idiosyncratic problems, the extrapolation of animal test results to humans in drug development, and the contribution of genetic variation in human P450s to cancer incidence.
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            Adaptation of the human aryl hydrocarbon receptor to sense microbiota-derived indoles

            Ligand activation of the aryl hydrocarbon (AHR) has profound effects upon the immunological status of the gastrointestinal tract, establishing and maintaining signaling networks, which facilitate host-microbe homeostasis at the mucosal interface. However, the identity of the ligand(s) responsible for such AHR-mediated activation within the gut remains to be firmly established. Here, we combine in vitro ligand binding, quantitative gene expression, protein-DNA interaction and ligand structure activity analyses together with in silico modeling of the AHR ligand binding domain to identify indole, a microbial tryptophan metabolite, as a human-AHR selective agonist. Human AHR, acting as a host indole receptor may exhibit a unique bimolecular (2:1) binding stoichiometry not observed with typical AHR ligands. Such bimolecular indole-mediated activation of the human AHR within the gastrointestinal tract may provide a foundation for inter-kingdom signaling between the enteric microflora and the immune system to promote commensalism within the gut.
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              Microbiome-derived tryptophan metabolites and their aryl hydrocarbon receptor-dependent agonist and antagonist activities.

              The tryptophan metabolites indole, indole-3-acetate, and tryptamine were identified in mouse cecal extracts and fecal pellets by mass spectrometry. The aryl hydrocarbon receptor (AHR) agonist and antagonist activities of these microbiota-derived compounds were investigated in CaCo-2 intestinal cells as a model for understanding their interactions with colonic tissue, which is highly aryl hydrocarbon (Ah)-responsive. Activation of Ah-responsive genes demonstrated that tryptamine and indole 3-acetate were AHR agonists, whereas indole was an AHR antagonist that inhibited TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)-induced CYP1A1 expression. In contrast, the tryptophan metabolites exhibited minimal anti-inflammatory activities, whereas TCDD decreased phorbol ester-induced CXCR4 [chemokine (C-X-C motif) receptor 4] gene expression, and this response was AHR dependent. These results demonstrate that the tryptophan metabolites indole, tryptamine, and indole-3-acetate modulate AHR-mediated responses in CaCo-2 cells, and concentrations of indole that exhibit AHR antagonist activity (100-250 μM) are detected in the intestinal microbiome.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                3 May 2016
                2016
                : 11
                : 5
                : e0154629
                Affiliations
                [1 ]INSERM, U1183, Institute of Regenerative Medicine and Biotherapy, Montpellier, F-34290, France
                [2 ]Montpellier University, UMR 1183, Montpellier, F-34203, France
                [3 ]Department of Food Science, Aarhus University, Foulum, Denmark
                [4 ]Institut de Recherche en Cancérologie de Montpellier, Montpellier, F-34298, France
                [5 ]CHU Montpellier, Institute of Regenerative Medicine and Biotherapy, Montpellier, F-34290, France
                French National Centre for Scientific Research, FRANCE
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: MKR PB BE MDC SGC. Performed the experiments: MKR PB SGC. Analyzed the data: MKR PB MDC SGC. Contributed reagents/materials/analysis tools: MKR PB SGC. Wrote the paper: MKR PB BE MDC SGC.

                Author information
                http://orcid.org/0000-0002-1889-2874
                Article
                PONE-D-15-54900
                10.1371/journal.pone.0154629
                4854444
                27138278
                c36eec66-5589-4759-b752-d621e723b2da
                © 2016 Rasmussen et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 4 January 2016
                : 16 April 2016
                Page count
                Figures: 7, Tables: 3, Pages: 17
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100008398, Villum Fonden (DK);
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003554, Lundbeckfonden (DK);
                Award Recipient :
                Funded by: Norma og Frode S Jacobsens Fond
                Award Recipient :
                This work was supported by Willum Fonden ( http://veluxfoundations.dk/en/forskning/teknisk-og-naturvidenskabelig-forskning), MKR; Lundbeckfonden ( http://www.lundbeckfoundation.com/), MKR; and Norma of Frode S Jacobsens Fond ( http://www.normaogfrodejacobsensfond.dk/menu.htm), MKR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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