Discovery and biological characterization of 1-(1H-indol-3-yl)-9H-pyrido[3,4-b]indole as an aryl hydrocarbon receptor activator generated by photoactivation of tryptophan by sunlight

Abstract

Activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is required for AHR dependent transcriptional activation and TCDD toxicity. We previously reported that aqueous tryptophan exposed to sunlight through window glass (aTRP) contains multiple photoproducts, including the well characterized 6-formylindolo[3,2-b]carbazole (FICZ), capable of activating the AHR and inducing CYP1A and CYP1A-mediated enzyme activities. We report here the isolation from aTRP and chemical characterization and synthesis of 1-(1H-indol-3-yl)-9H-pyrido[3,4-b]indole (IPI), a compound previously identified as a natural product of marine ascidia and now shown to be a TRP photoproduct with AHR-inducing properties. IPI, FICZ and TCDD produced equieffective induction of CYP1A-mediated 7-ethoxyresorufin deethylase (EROD) activity in chick embryo primary hepatocytes and mammalian Hepa1c1c7 cells. EROD induction by IPI was markedly curtailed in AHR-defective c35 cells, supporting the AHR dependence of the IPI response. Although IPI had a higher EC₅₀ for EROD induction than FICZ, the much larger amount of IPI than FICZ in aTRP makes IPI a prominent contributor to EROD induction in aTRP. IPI was detected in TRP-containing culture medium under ambient laboratory conditions but not in TRP-free medium, consistent with its production from TRP. Cotreatment of hepatocytes with submaximal EROD-inducing doses of IPI and FICZ or TCDD produced additive increases in EROD without synergistic or inhibitory interactions. IPI and FICZ were readily metabolized by cultured hepatocytes. In addition to increasing CYP1A4 mRNA and EROD, IPI and FICZ decreased hepatocyte phosphoenolpyruvate carboxykinase mRNA expression and glucose output, biological effects associated with TCDD metabolic dysregulation. The findings underscore a role for sunlight in generating AHR-activating bioactive molecules.

Introduction

The environmental toxin and industrial by-product TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin, dioxin) is the most potent and best studied activator of the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor and member of the Per-Arnt-Sim (PAS) protein family [1], [2]. Activation of the AHR by TCDD elicits diverse pathological effects, including tumor promotion and metabolic dysregulation leading to a wasting syndrome [1], [2], [3].

Ligand activation causes the AHR to migrate from cytosol to the nucleus and form a complex with the aryl hydrocarbon nuclear translocator (ARNT) that can bind dioxin-responsive elements (DREs) in the promoter regions of target genes including xenobiotic-metabolizing cytochrome P450 (CYP1A) enzymes and TCDD-inducible poly [ADP-ribose] polymerase (TiPARP, PARP7) [4]. In addition to environmental toxins like TCDD, some natural products have recently been shown to activate the AHR, among which tryptophan (TRP) derivatives comprise a large group [5]. These include TRP derivatives ingested in the diet or produced by metabolism in vivo, such as indolo[3,2-b]carbazole (ICZ) [6], and photoproducts formed by exposure of TRP to UV or visible light [7], [8], [9], among which 6-formylindolo[3,2-b]carbazole (FICZ) is the best characterized [10]. The discovery of natural compounds that can bind and activate the AHR has stimulated a search for diverse AHR ligands that may elicit pathologic effects (e.g., tumorigenesis, metabolic dysregulation) or as yet unrecognized physiologic activities of the AHR.

We previously reported that exposure of an aqueous solution of TRP to sunlight passing through window glass (aTRP) produces multiple photoproducts capable of activating the AHR and inducing CYP1A-mediated enzyme activities, including arachidonic acid epoxygenation and 7-ethoxyresorufin deethylation (EROD) [8]. FICZ was identified in one of 14 fractions obtained by separation of aTRP by reverse phase high pressure liquid chromatograph (RP-HPLC), all of which exhibited CYP1A inducing capacity. That prior study showed that photoactivated TRP contains many AHR inducers in addition to FICZ. We report here the identification, chemical characterization and synthesis of a novel photoproduct present in aTRP that induces CYP1A with high efficacy: 1-(1H-indol-3-yl)-9H-pyrido[3,4-b]indole (IPI).