Abstract
Dioxins and other polycyclic aromatic compounds formed during the combustion of waste and fossil fuels represent a risk to human health, as well as to the well being of our environment. Compounds of this nature exert carcinogenic and endocrine-disrupting effects in experimental animals by binding to the orphan aryl hydrocarbon receptor (AhR). Understanding the mechanism of action of these pollutants, as well as the physiological role(s) of the AhR, requires identification of the endogenous ligand(s) of this receptor. We reported earlier that activation of AhR by ultraviolet radiation is mediated by the chromophoric amino acid tryptophan (Trp), and we suggested that a new class of compounds derived from Trp, in particular 6-formylindolo[3,2-b]carbazole (FICZ), acts as natural high affinity ligands for this receptor. Here we describe seven new FICZ-derived indolo[3,2-b]carbazole-6-carboxylic acid metabolites and two sulfoconjugates, and we demonstrate the following. (i) FICZ is formed efficiently by photolysis of Trp upon exposure to visible light. (ii) FICZ is an exceptionally good substrate for cytochromes P450 (CYP) 1A1, 1A2, and 1B1, and its hydroxylated metabolites are remarkably good substrates for the sulfotransferases (SULTs) 1A1, 1A2, 1B1, and 1E1. Finally, (iii) sulfoconjugates of phenolic metabolites of FICZ are present in human urine. Our findings indicate that formylindolo[3,2-b]carbazols are the most potent naturally occurring activators of the AhR signaling pathway and may be the key substrates of the CYP1 and SULT1 families of enzymes. These conclusions contradict the widespread view that xenobiotic compounds are the major AhR ligands and CYP1 substrates.
Highlights
The aryl hydrocarbon receptor (AhR) protein has been shown to bind the compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as well as similar highly lipophilic halogenated and non-halogenated hydrocarbons leading to cardiovascular, carcinogenic, and endocrine effects [12,13,14,15,16]
Per unit of energy, UVB was the most efficient in generating FICZ. 40 – 400-Fold increases in FICZ formation were observed in the presence of the photosensitizer riboflavin, especially in the case of UVA and visible light because, in contrast to Trp, riboflavin absorbs light
6a 2-OH-CICZ 6b OH-CICZ* 6c 8-OH-CICZ 7a 2,8-dOH-CICZ 7b 2,10-dOH-CICZ. Efficiently at these longer wavelengths. This relatively easy conversion of Trp to FICZ in the presence of riboflavin and light suggests that the same process could occur, e.g. in the skin, and that the formation of FICZ could probably explain the reported UV-dependent activation of CYP1 enzymes in human skin [32]
Summary
The AhR protein has been shown to bind the compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as well as similar highly lipophilic halogenated and non-halogenated hydrocarbons leading to cardiovascular, carcinogenic, and endocrine effects [12,13,14,15,16]. It seems likely that for purposes of regulation, endogenous ligands of AhR are metabolized rapidly, so that the use of persistent xenobiotics such as dioxins to investigate this receptor might be inappropriate. Many endogenous compounds, including various indoles, heme, and arachidonic acid metabolites, have been found to be AhR agonists, and Trp is the precursor for many of the most active ligands [3]. FICZ up-regulates the expression of AhR-responsive genes efficiently and transiently and is rapidly metabolized in a feedback manner [23]. This sequence of events typical for autoregulatory loops in biological signaling indicates that this compound may be a physiological ligand for AhR. The purpose of this work was to characterize the light-dependent formation of FICZ, its metabolism by human enzymes, and to document the presence of stable metabolites of FICZ in humans
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