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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
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
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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.

Place, publisher, year, edition, pages
2005. Vol. 85, no 2, 935-943 p.
Keyword [en]
tryptophan; 6-formylindolo[3, 2-b]carbazole; light; aryl hydrocarbon receptor.
URN: urn:nbn:se:su:diva-23544DOI: 10.1093/toxsci/kfi154OAI: diva2:192694
Part of urn:nbn:se:su:diva-341Available from: 2005-01-27 Created: 2005-01-27 Last updated: 2010-08-11Bibliographically approved
In thesis
1. 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
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)
Available from: 2005-01-27 Created: 2005-01-27 Last updated: 2010-01-11Bibliographically approved

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