Naturally occurring marine brominated indoles are aryl hydrocarbon receptor ligands/agonists

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the toxic and biological effects of structurally diverse chemicals, including the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). As part of a larger effort to identify the full spectrum of chemicals that can bind to and activate the AhR, we have examined the ability of several naturally occurring marine-derived brominated indoles and brominated (methylthio)indoles (collectively referred to as brominated indoles) to bind to the AhR and stimulate AhR-dependent gene expression. Incubation of mouse, rat, and guinea pig recombinant cell lines containing a stably transfected AhR-responsive luciferase reporter gene with eight brominated indoles revealed that all compounds stimulated luciferase reporter gene activity, although some species-specific differences were observed. All compounds induced significantly more luciferase activity when incubated with cells for 4 h as compared to 24 h, demonstrating that these compounds are transient activators of the AhR signaling pathway. Three of the brominated indoles induced CYP1A1 mRNA in human HepG2 cells in vitro and Cyp1a mRNA in zebrafish embryos in vivo. The identification of the brominated indoles as direct ligands and activators/agonists of the AhR was confirmed by their ability to compete with [(3)H]TCDD for binding to the AhR and to stimulate AhR transformation and DNA binding in vitro. Taken together, these results indicate that marine-derived brominated indoles are members of a new class of naturally occurring AhR agonists.

Figure 1

Structures of the eight naturally-occurring, marine-derived brominated indoles. Abbreviations used: JN2-9-1: 2,4,6-tribromo-3-(methylthio)indole JN2-10-21: 2,3,4,6-tetrabromoindole JN2-11-1: 6-bromo-2,3-bis(methylthio)indole JN2-11-2: 2,4,5,6-tetrabromo-3-(methylthio)indole JN2-13-1: 4,5,6-tribromo-2-(methylthio)indole JN2-18-4: 4,5,6-tribromo-2,3-bis(methylthio)indole JN2-36-12: 4,6-dibromo-2-(methylsulfinyl)-3-(methylthio)indole (Itomanindole A) JN2-39-2: 3-acetyl-4,6-dibromo-2,3-dihydro-1H-indole-3-carboxylic acid.

Figure 2

Brominated indoles are weak activators of AhR-dependent CYP1A expression in PLHC-1 cells. Teleost fish hepatoma (PLHC-1) cells were incubated with DMSO (0.5%), TCDD (0.1 nM) or each brominated indole (10 μM) for 24 h after which ethoxyresorufin O-deethylase (EROD) activity was measured and the results normalized to protein content. Results are presented as the percent of the maximum EROD induction by TCDD, and are the mean ± SE of at least triplicate determinations.

Figure 3

Brominated indoles stimulate AhR-dependent luciferase reporter gene expression in mammalian cells. Recombinant mouse and rat hepatoma (H1L1.1c2 and H4L1.1c4, respectively) and guinea pig intestinal adenocarcinoma (G16L1.1c8) cells were incubated with DMSO (1%), TCDD (1 nM) or the indicated brominated indole (10 μM) for 4 h after which luciferase activity was determined. Values are expressed as a mean percent ± SD of the maximal induction resulting from 1 nM TCDD in triplicate incubations. A decrease in luciferase activity in the H1L1.1c2 cell line compared to both the G16L1.1c8 and H4L1.1c4 cell lines is indicated (*p < 0.005 (for both comparisons); Student’s t-test). Results are representative of duplicate independent experiments.

Figure 4

Brominated indoles are transient activators of the AhR signaling pathway. Recombinant rat hepatoma cells (H4L1.1c4) were incubated with DMSO (1%), TCDD (1 nM) or the indicated brominated indole (10 μM) for 4 and 24 h after which luciferase activity was determined. Values are expressed as an average of relative light units (RLU) ± SD of triplicate incubations. Results are representative of duplicate independent experiments.

Figure 5

Brominated indoles stimulate mammalian AhR transformation and DNA binding in vitro. Guinea pig hepatic cytosol (8 mg protein/ml) was incubated with DMSO, TCDD (20 nM) and each brominated indole (64 μM) for 2 h at 20°C followed by incubation with [³²P]DRE oligonucleotide. Protein-DNA complexes were resolved by EMSA and visualized by autoradiography. The arrow indicates the position of the inducible AhR-DNA complex.

Figure 6

Brominated indoles competitively inhibit the specific binding of [³H]TCDD to mouse hepatic cytosolic AhR. Cytosolic extracts (1 mg protein/ml) prepared from mouse hepatoma cells (Hepa1c1c7) were incubated with 1 nM [³H]TCDD, in the absence (●) or presence ( ) of 0.2 μM TCDF, or 5 μM of each brominated indole for 1 h on ice. Aliquots (300 μl) of each incubation were analyzed for ligand binding by sucrose density gradient centrifugation.

Figure 7

Brominated indoles induce Cyp1a mRNA in zebrafish embryos in vivo. Zebrafish embryos (48 hpf) were exposed to DMSO (0.5%), TCDD (2 nM), or each of three indicated brominated indoles (2 μM) for 6 hr, after which RNA was isolated and Cyp1a mRNA was measured by real time RT-PCR. Values represent the average relative expression of Cyp1a mRNA ± SD of triplicate samples. Each sample was a pool of 20 embryos. Cyp1A expression was normalized to that of beta-actin.

Figure 8

Brominated indoles are transient inducers of CYP1A1 mRNA in human cells. HepG2 cells were incubated at 37°C with DMSO (0.5%), TCDD (2 nM), or each of three indicated brominated indoles (10 μM) for 6 or 24 hr, after which RNA was isolated and CYP1A1 mRNA was measured by real time RT-PCR. Values represent the average relative expression of CYP1A1 mRNA ± SD of triplicate samples. CYP1A1 expression was normalized to that of beta-actin.