Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates genes involved in xenobiotic metabolism, cellular proliferation, and differentiation. Numerous xenobiotic and biological compounds are known to interact with AhR, but it remains an orphan receptor, because its physiological ligand is unknown. We identified AhR ligands in human urine using a yeast AhR signaling assay and then characterized their properties. Two ligands, indirubin and indigo, were both present at average concentrations of ϳ0.2 nM in the urine of normal donors. Indirubin was also detected in fetal bovine serum and contributed half of the total AhR ligand activity. The activities of indirubin and indigo were comparable with or more potent than that of the archetypal ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, in yeast AhR activation assays. We suggest that the endogenous levels and potencies of indirubin and indigo are such that they activate AhR-mediated signaling mechanisms in vivo.
AhR,1 also called the dioxin receptor, is a ligand-activated transcription factor that is present in most cell and tissue types of the body (1). AhR-mediated signaling is required for potent xenobiotic ligands such as TCDD and polychlorinated biphenyls to produce toxic responses (2, 3). Toxic effects that are linked to xenobiotic AhR ligand exposures in animals include cancers, reproductive impairment, endometriosis, birth defects, and immunological impairment (4 -6). The toxic potential of xenobiotic AhR ligands is currently a major concern for regulatory agencies that are responsible for protecting public and environmental health. Although numerous xenobiotic ligands for AhR have been identified, the AhR is considered to be an orphan receptor, because its physiological ligand(s) and its function are not known. Tryptophan and other indole-containing compounds (7-9), bilirubin (10), 7-ketocholesterol (11), lipoxin A4 (12), flavones, and related compounds (13) interact with AhR to produce activation or inhibition of signal transduction. In general, the low levels, lack of potency, and restricted distribution of these compounds make them unlikely candidates as major regulators of AhR signaling in most tissue types. We reasoned that human urine might be a good place to search for endogenous AhR ligands, and we developed a methodology to detect and isolate such compounds.
EXPERIMENTAL PROCEDURESMaterials-Blue rayon was kindly provided by Dr. Hayatsu (Okayama University, Okayama City, Japan). General chemicals, essentially analytical grade, were purchased from Wako (Kyoto, Japan). TCDD was purchased from CIL (Andover, MA, USA). Indigo and -glucuronidase were purchased from Sigma. Indirubin was synthesized as described in Hoessel et al. (14). Indigo and indirubin were further purified by HPLC before use.Yeast Assay for AhR Ligand Activity-The assay procedure was essentially as described by Miller (15). The yeast strain YCM3 was grown overnight at 30°C in synthetic glucose medium lacking tryptophan. Test chemicals (dissolved in Me 2 SO...
