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Metabolic fate of the Ah receptor ligand 6-formylindolo[3,2-b]carbazole
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
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2004 (English)In: Chemico-Biological Interactions, ISSN 0009-2797, Vol. 149, no 2-3, 151-164 p.Article in journal (Refereed) Published
Abstract [en]

The physiological role of the aryl hydrocarbon receptor (AhR), a member of the basic helix-loop-helix PER-ARNT-SIM (PAS) transcription factor family is not known. We have suggested that the AhR is involved in light signaling through binding of photoproducts with high AhR affinity. This suggestion is based on (i) the high AhR affinity of the tryptophan photoproduct formylindolo[3,2-b]carbazole (FICZ), (ii) the induction of rapid and transient expression of AhR-regulated genes by FICZ and by extracts of UV-irradiated tryptophan as well as (iii) the fact that light induces the AhR-regulated cytochrome P450s CYP1A1, CYP1B1 and CYP2S1. The transient mRNA expression caused by light and tryptophan photoproducts suggests that the biotransformation enzymes induced by AhR activation take part in a metabolic degradation of the natural AhR ligand. This study aimed at identifying the involvement of phase I and phase II enzymes in the metabolic degradation of FICZ. A cytochrome P450-dependent metabolism of FICZ giving rise to preferentially mono- and di-hydroxylated derivatives has earlier been reported. In the present study, rat and human hepatic S9 mixes were employed together with specific enzyme inhibitors and cofactors. Compared to the Aroclor-induced rat liver S9, the non-induced rat liver S9 and the human liver S9 caused a more complex metabolite profile of FICZ. The CYP1A1 enzyme was confirmed to be the most important enzyme for the first step in the metabolism. CYP1A2 was found to have overlapping specificity with CYP1A1 being able to form the same major metabolites although with different kinetics. CYP1B1 turned out to be preferentially involved in the further metabolism of dihydroxylated metabolites. Microsomal epoxide hydrolase, and as yet not identified forms of sulphotransferases and glucuronosyltransferases were also found to take part in the metabolic degradation of FICZ. Thus, tryptophan photoproducts fit into a model in which the ligand-activated AhR signaling is autoregulated by the induced metabolic enzymes.

Place, publisher, year, edition, pages
2004. Vol. 149, no 2-3, 151-164 p.
Keyword [en]
Animals, Aroclor 1254/metabolism, Carbazoles/*metabolism/toxicity, Chromatography; High Pressure Liquid, Cytochrome P-450 Enzyme System/antagonists & inhibitors/*metabolism, Enzyme Inhibitors/pharmacology, Humans, Indoles/*metabolism/toxicity, Male, Microsomes; Liver/enzymology/*metabolism, Rats, Rats; Sprague-Dawley, Receptors; Aryl Hydrocarbon/*metabolism, Tryptophan/metabolism
Identifiers
URN: urn:nbn:se:su:diva-16046DOI: 10.1016/j.cbi.2004.08.005PubMedID: 15501436OAI: oai:DiVA.org:su-16046DiVA: diva2:182566
Available from: 2008-12-12 Created: 2008-12-12 Last updated: 2010-01-11Bibliographically approved
In thesis
1. Direct and indirect mechanisms for aryl hydrocarbon receptor activation mediated by 6-formylindolo[3,2-b]carbazole
Open this publication in new window or tab >>Direct and indirect mechanisms for aryl hydrocarbon receptor activation mediated by 6-formylindolo[3,2-b]carbazole
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aryl hydrocarbon receptor (AhR) is mostly recognized for mediating the adverse effects of dioxins. In addition, endogenous activation of the AhR seems to have important biological functions.

 Several studies have demonstrated an activation of the receptor when no exogenous ligand was added. Furthermore, different physical stimuli such as UV irradiation, fluid shear stress, and hyperoxia have been shown to induce AhR-dependent transcriptional activity. Together these reports indicate either the presence of endogenous ligands or a non-ligand dependent activation. While the mechanisms behind such responses are still elusive, formation of tryptophan photoproducts with high AhR-affinity has been suggested to explain the activation observed after UVB irradiation. The photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) has been proposed to be an endogenous signal substance, and is the focus of the present thesis.

 The objective of the work presented in this thesis was to further characterize the formation and metabolism of FICZ, to identify the biotransforming enzymes required for its metabolism, and subsequently to isolate FICZ-derived metabolites in human urine.

 The studies reveal that FICZ is an excellent substrate for CYP1 enzymes resulting in an efficient metabolism and rapid clearance of FICZ, and a reduced or abolished affinity for the AhR. The hydroxylated metabolites are in turn very good substrates for sulfo-conjugation, and monosulfated derivatives of FICZ were identified in human urine, proving the existence of FICZ in vivo. Furthermore, disturbing the CYP1-dependent metabolic clearance of FICZ efficiently attenuated the rapid depletion of intracellular levels of FICZ, and resulted in a delayed and prolonged AhR-activation. These results suggest that inhibition of degradation of FICZ provides a potent mechanism for indirect regulation of the AhR response.

 The high affinity and AhR activating capacity, together with its rapid clearance by AhR regulated biotransforming enzymes and presence in humans in vivo, all strengthen the hypothesis that FICZ is an endogenous ligand for the AhR and an important biological signaling molecule.

 

Place, publisher, year, edition, pages
Stockholm: Department of Genetics, Microbiology and Toxicology, Stockholm University, 2009. 49 p.
National Category
Pharmacology and Toxicology
Research subject
Genetic Toxicology
Identifiers
urn:nbn:se:su:diva-29266 (URN)978-91-7155-926-5 (ISBN)
Public defence
2009-09-18, G-salen, Svante Arrhenius väg 20 C, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: In progress.Available from: 2009-08-27 Created: 2009-08-19 Last updated: 2009-08-20Bibliographically approved
2. Formation and metabolism of the tryptophan-derived 6-formylindolo[3,2-b]carbazole - a light-induced Ah-receptor ligand
Open this publication in new window or tab >>Formation and metabolism of the tryptophan-derived 6-formylindolo[3,2-b]carbazole - a light-induced Ah-receptor ligand
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aryl hydrocarbon receptor (AhR) is a ligand dependent transcription factor ubiquitously expressed in mammalian cells. It is a genetically ancient protein mostly known for binding the extremely toxic contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Binding to the AhR explains the variety of toxic responses of TCDD as well as the induction of several drug metabolizing enzymes. Induction of cytochrome P4501A1 (CYP1A1) is the most well characterized of the AhR regulated responses. The physiological functions of AhR and the endogenous ligand(s) for the receptor are under investigation but are not yet unraveled.

Several tryptophan (TRP) derived indol-containing compounds have been reported to possess AhR affinity/CYP1A1 inducing capacity and TRP mediates CYP1A1 induction by UV light. The TRP photoproduct, 6-formylindolo[3,2-b]carbazole (FICZ) has the highest AhR affinity described so far and it causes a rapid and transient induction of the CYP1A1 gene in human cells. A number of reports on constitutive CYP1A1 activity in cultured cells is therefore most likely explained by the presence of TRP-derived AhR ligands in cell culture media.

The aims of the studies were to investigate the impact of FICZ and FICZ metabolism on CYP1A1 gene regulation, to explore the metabolic fate of FICZ and to identify whether normal laboratory light could lead to formation of FICZ and thereby contribute to earlier observed CYP1A1 inducing effects by cell culture media.

Metabolic studies using fractions of Aroclor-induced and non-induced rat liver and human liver as well as heterologously expressed enzymes revealed that FICZ can be efficiently metabolized by the CYP enzymes 1A1 and 1A2 and by an unknown cytosolic enzyme, to a number of hydroxylated and other oxidized metabolites. All of the hitherto identified 11 hydroxylated metabolites of FICZ are prone to conjugation reactions by glucuronosyltranferases and sulfotransferases. The metabolites formed by human enzymes are primarily sulfated. Thus, the sulfated metabolites of FICZ will be crucial in the future analyzes of FICZ formation in vivo. FICZ was identified to be formed, not only by UV illumination, but also by normal laboratory light. The constitutive CYP1A1 activity was significantly induced through the formation of several TRP related photoproducts in light-exposed medium. One of these photoproducts was identified as FICZ. Thus, the TRP photoproduct, FICZ, fits into a model in which FICZ auto-regulates the expression of induced enzymes. It is hypothesized that FICZ might function as a chemical messenger that activates AhR in response to light and might be one of several possible endogenous AhR ligands.

Place, publisher, year, edition, pages
Stockholm: Institutionen för genetik, mikrobiologi och toxikologi, 2005. 58 p.
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:su:diva-341 (URN)91-7265-993-9 (ISBN)
Public defence
2005-02-18, De Geersalen, Geovetenskapens hus, Svante Arrhenius väg 8 A, Stockholm, 13:00 (English)
Supervisors
Available from: 2005-01-27 Created: 2005-01-27 Last updated: 2010-01-11Bibliographically approved

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