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The impact of cytochrome P4501-inhibitors on aryl hydrocarbon receptor signaling
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aryl hydrocarbon receptor (AHR) best known as a ligand-activated transcription factor that mediates toxic responses to xenobiotics such as dioxins, is also activated by certain endogenous compounds. Activation of the AHR up-regulates transcription of a large number of genes, including those encoding members of the cytochrome P450 1 family of enzymes (CYP1s). Although the AHR has been shown to be involved in several normal processes, its physiological role remains elusive. The endogenous ligand 6-formylindolo[3,2-b]carbazole (FICZ), formed from tryptophan, is present in cell culture media and biological specimens. FICZ is an excellent substrate for CYP1 enzymes and together FICZ/AHR/CYP1A1 interactions constitute an auto regulatory feedback loop that controls AHR signaling. A vast number of compounds that inhibit CYP1 enzymes have been reported to be AHR activators, even though they have little or no affinity for the receptor. We hypothesized, that their agonistic effects are dependent on the presence of background levels of FICZ. To test this, AHR signaling in different cell systems exposed to FICZ and/or inhibitors was assessed by measuring EROD activity and CYP1A1 transcription. In addition to a commercial culture medium, a medium free of background levels of FICZ was used. Activation of AHR by of a diverse set of CYP1A1 inhibitors did require FICZ in the culture medium. Furthermore, the compounds tested both prolonged and potentiated FICZ-induced receptor signaling. On the basis of these observations we propose that a compound may activate AHR signaling indirectly by inhibiting CYP1A1 and thereby attenuating the metabolism of FICZ. This mechanism was confirmed for certain polyphenols and pharmaceuticals. Surprisingly, the activating capacity and potentiating effect of two pharmaceuticals on AHR signaling could not be explained by the mechanism proposed, and we speculated that in these cases the agonistic effect might involve interactions of the cellular antioxidant response with the basic transcription machinery. Together, our observations provide a mechanistic explanation as to how compounds that inhibit CYP1A1 can activate AHR signaling. They also indicate that the general perception of the binding pocket of AHR as promiscuous, is probably wrong. The fact that indirect activation of AHR may cause sustained signaling requires further studies in vivo not least, in order to prevent toxicity.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2016. , 52 p.
National Category
Cell and Molecular Biology
Research subject
Molecular Bioscience
Identifiers
URN: urn:nbn:se:su:diva-128367ISBN: 978-91-7649-385-4 (print)OAI: oai:DiVA.org:su-128367DiVA: diva2:914513
Public defence
2016-05-13, Atrium, Nobels väg 12 B, Karolinska Institutet, Solna, 10:00 (Swedish)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

Available from: 2016-04-20 Created: 2016-03-24 Last updated: 2017-02-20Bibliographically approved
List of papers
1. Inhibition of cytochrome P4501-dependent clearance of the endogenous agonist FICZ as a mechanism for activation of the aryl hydrocarbon receptor
Open this publication in new window or tab >>Inhibition of cytochrome P4501-dependent clearance of the endogenous agonist FICZ as a mechanism for activation of the aryl hydrocarbon receptor
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2012 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 12, 4479-4484 p.Article in journal (Refereed) Published
Abstract [en]

Altered systemic levels of 6-formylindolo[3,2-b]carbazole (FICZ), an enigmatic endogenous ligand for the aryl hydrocarbon receptor (AHR), may explain adverse physiological responses evoked by small natural and anthropogenic molecules as well as by oxidative stress and light. We demonstrate here that several different chemical compounds can inhibit the metabolism of FICZ, thereby disrupting the autoregulatory feedback control of cytochrome P4501 systems and other proteins whose expression is regulated by AHR. FICZ is both the most tightly bound endogenous agonist for the AHR and an ideal substrate for cytochrome CYP1A1/1A2 and 1B1, thereby also participating in an autoregulatory loop that keeps its own steady-state concentration low. At very low concentrations FICZ influences circadian rhythms, responses to UV light, homeostasis associated with pro-and anti-inflammatory processes, and genomic stability. Here, we demonstrate that, if its metabolic clearance is compromised, femtomolar background levels of this compound in cell-culture medium are sufficient to up-regulate CYP1A1 mRNA and enzyme activity. The oxidants UVB irradiation and hydrogen peroxide and the model AHR antagonist 3'-methoxy-4'-nitroflavone all inhibited induction of CYP1A1 enzyme activity by FICZ or 2,3,7,8-tetrachlorodibenzo-p-dioxin, thereby subsequently elevating intracellular levels of FICZ and activating AHR. Taken together, these findings support an indirect mechanism of AHR activation, indicating that AHR activation by molecules with low affinity actually may reflect inhibition of FICZ metabolism and raising questions about the reported promiscuity of the AHR. Accordingly, we propose that prolonged induction of AHR activity through inhibition of CYP1 disturbs feedback regulation of FICZ levels, with potential detrimental consequences.

National Category
Biochemistry and Molecular Biology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-76144 (URN)10.1073/pnas.1118467109 (DOI)000301712600032 ()
Note

7

Available from: 2012-05-09 Created: 2012-05-09 Last updated: 2017-12-07Bibliographically approved
2. Quercetin, Resveratrol, and Curcumin Are Indirect Activators of the Aryl Hydrocarbon Receptor (AHR)
Open this publication in new window or tab >>Quercetin, Resveratrol, and Curcumin Are Indirect Activators of the Aryl Hydrocarbon Receptor (AHR)
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2012 (English)In: Chemical Research in Toxicology, ISSN 0893-228X, E-ISSN 1520-5010, Vol. 25, no 9, 1878-1884 p.Article in journal (Refereed) Published
Abstract [en]

Several polyphenols have been shown to activate the aryl hydrocarbon receptor (AHR) in spite of the fact that they bind to the receptor with low affinity. The aim of this study was to investigate whether quercetin (QUE), resveratrol (RES), and curcumin (CUR) interfere with the metabolic degradation of the suggested endogenous AHR ligand 6-formylindolo[3,2-b]carbazole (FICZ) and thereby indirectly activate the AHR. Using recombinant human enzyme, we confirmed earlier reported inhibitory effects of the polyphenols on cytochrome P4501A1 (CYP1A1) activity, and inhibition of metabolic clearance of FICZ was documented in FICZ-treated immortalized human keratinocytes (HaCaT). CYP1A1 activity was induced in HaCaT cells by all three compounds, and when they were added together with FICZ, a prolonged activation was observed after a dose-dependent inhibition period. The same pattern of responses was seen at the transcriptional level as determined with a CYP1A1 reporter assay in human liver hepatoma (HepG2) cells. To test the ability of the polyphenols to activate the AHR in the absence of FICZ, the cells were treated in medium, which in contrast to commercial batches of medium did not contain background levels of FICZ. Importantly, AHR activation was only observed in the commercial medium. Taken together, these findings suggest that QUE, RES, and CUR induce CYP1A1 in an indirect manner by inhibiting the metabolic turnover of FICZ. Humans are exposed to these compounds through the diet and nutritional supplements, and we propose that altered systemic levels of FICZ caused by such compounds may have physiological consequences.

National Category
Biomedical Laboratory Science/Technology Pharmacology and Toxicology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-81821 (URN)10.1021/tx300169e (DOI)000308777100009 ()
Note

AuthorCount:5;

Available from: 2012-11-07 Created: 2012-11-01 Last updated: 2017-12-07Bibliographically approved
3. Ketoconazole, omeprazole, and primaquine prolong and enhance the aryl hydrocarbonreceptor signaling induced by the endogenous ligand FICZ
Open this publication in new window or tab >>Ketoconazole, omeprazole, and primaquine prolong and enhance the aryl hydrocarbonreceptor signaling induced by the endogenous ligand FICZ
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Several compounds that inhibit cytochrome P4501 (CYP1) enzymes have been shown to actas aryl hydrocarbon receptor (AHR) agonists by reducing the metabolic turnover of the endogenous receptor ligand 6-formylindolo[3,2-b]carbazole (FICZ). In this study we aimed at investigating whether a group of widely prescribed drugs, namely ketoconazole (KTZ),omeprazole (OME) and primaquine (PQ), can act as indirect AHR activators via this mechanism. Inhibitory effects of KTZ, PQ, and OME were measured in CYP1A1 expressing supersomes and all three drugs inhibited CYP1A1 activity. KTZ was the most efficient inhibitor and HPLC analysis revealed that KTZ slowed down the metabolic turnover of intracellular FICZ. All three drugs induced the catalytic activity of CYP1A1 (7-ethoxyresorufin-O-deethylase, EROD) in HaCaT cells as well as increased the expression of CYP1A1 mRNA. Co-exposure to the drugs with FICZ prolonged and enhanced FICZ-induced AHR activation in a synergistic manner. Our findings indicate that KTZ activate AHR by inhibiting the metabolic turnover of FICZ. Interestingly, PQ and OME seem to act by other mechanisms that sensitize the cells to FICZ-dependent transcriptional activation of the AHR.To the author’s knowledge, this is the first publication indicating that KTZ, OME, and PQ can superinduce AHR signaling by increasing the responses to an endogenous receptor ligand.

National Category
Biological Sciences
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-128366 (URN)
Available from: 2016-03-24 Created: 2016-03-24 Last updated: 2016-03-30Bibliographically approved

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