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Formation and metabolism of the tryptophan-derived 6-formylindolo[3,2-b]carbazole - a light-induced Ah-receptor ligand
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
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: urn:nbn:se:su:diva-341ISBN: 91-7265-993-9 (print)OAI: oai:DiVA.org:su-341DiVA: diva2:192695
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
List of papers
1. Regulation of CYP1A1 transcription via the metabolism of the tryptophan-derived 6-formylindolo[3,2-b]carbazole
Open this publication in new window or tab >>Regulation of CYP1A1 transcription via the metabolism of the tryptophan-derived 6-formylindolo[3,2-b]carbazole
2000 In: Archives of Biochemistry and Biophyics, ISSN 0003-9861, Vol. 383, no 1, 99-107 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-23540 (URN)
Note
Part of urn:nbn:se:su:diva-341Available from: 2005-01-27 Created: 2005-01-27Bibliographically approved
2. Characterization of in vitro metabolites of the aryl hydrocarbon receptor ligand 6-formylindolo[3,2-b]carbazole by liquid chromatography-mass spectrometry and NMR
Open this publication in new window or tab >>Characterization of in vitro metabolites of the aryl hydrocarbon receptor ligand 6-formylindolo[3,2-b]carbazole by liquid chromatography-mass spectrometry and NMR
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2003 In: Drug Metabolism and Dispositions, ISSN 0090-9556, Vol. 31, no 2, 233-241 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-23541 (URN)
Note
Part of urn:nbn:se:su:diva-341Available from: 2005-01-27 Created: 2005-01-27Bibliographically approved
3. Metabolic fate of the Ah receptor ligand 6-formylindolo[3,2-b]carbazole
Open this publication in new window or tab >>Metabolic fate of the Ah receptor ligand 6-formylindolo[3,2-b]carbazole
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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.

Keyword
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:nbn:se:su:diva-16046 (URN)10.1016/j.cbi.2004.08.005 (DOI)15501436 (PubMedID)
Available from: 2008-12-12 Created: 2008-12-12 Last updated: 2010-01-11Bibliographically approved
4. Predicting the in vivo metabolism of the AhR-ligand 6-formylindolo[3,2-b]carbazole employing human metabolic enzymes
Open this publication in new window or tab >>Predicting the in vivo metabolism of the AhR-ligand 6-formylindolo[3,2-b]carbazole employing human metabolic enzymes
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Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-23543 (URN)
Note
Part of urn:nbn:se:su:diva-341Available from: 2005-01-27 Created: 2005-01-27 Last updated: 2010-01-13Bibliographically approved
5. Identification of the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole in cell culture medium, as a factor that controls the constitutive aryl hydrocarbon receptor activity
Open this publication in new window or tab >>Identification of the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole in cell culture medium, as a factor that controls the constitutive aryl hydrocarbon receptor activity
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2005 (English)In: Toxicological Sciences, ISSN 1096-6080, E-ISSN 1096-0929, Vol. 85, no 2, 935-943 p.Article in journal (Refereed) Published
Abstract [en]

The presence of high affinity ligands for the aryl hydrocarbon receptor (AhR) in cell culture medium has generally been overlooked. Such compounds may confound mechanistic studies of the important AhR regulatory network. Numerous reports have described that light exposed cell culture medium induces AhR-dependent activity. In this study, we aimed at identifying the causative substance(s). A three-dimensional factorial design was used to study how the background activity of CYP1A1 in a rat hepatoma cell line (MH1C1) was controlled by photoproducts formed in the medium exposed to normal laboratory light. The light induced activity was found to be tryptophan dependent, but independent of riboflavin and other components in the medium. The light exposed medium showed the same transient enzyme inducing activity in vitro as the AhR ligand 6-formylindolo[3,2-b]carbazole (FICZ). This substance, which we have previously identified as being formed in UV-exposed tryptophan solutions, is a substrate for CYP1A1 and it has a higher AhR binding affinity than TCDD. Several tryptophan related photoproducts were detected in the light-exposed medium. For the first time one of the formed photoproducts was identified as FICZ with bioassay driven fractionation coupled with HPLC/MS. These results clearly show that tryptophan derived AhR ligands, which have been suggested to be endogenous AhR ligands, influence the background levels of CYP1A1 activity in cells in culture.

Keyword
tryptophan; 6-formylindolo[3, 2-b]carbazole; light; aryl hydrocarbon receptor.
Identifiers
urn:nbn:se:su:diva-23544 (URN)10.1093/toxsci/kfi154 (DOI)
Note
Part of urn:nbn:se:su:diva-341Available from: 2005-01-27 Created: 2005-01-27 Last updated: 2017-12-13Bibliographically approved

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