Responsiveness of a Xenopus laevis cell line to the aryl hydrocarbon receptor ligands 6-formylindolo[3,2-b]carbazole (FICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Abstract

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of environmental contaminants, such as 2, 3,7,. Frogs are very insensitive to TCDD toxicity, and AHRs from Xenopus laevis (African clawed frog) bind TCDD with >20-fold lower affinity than mouse AHR b-1 . Frog AHRs may nonetheless be highly responsive to structurally distinct compounds, especially putative endogenous ligands. We sought to determine the responsiveness of an X. laevis cell line, XLK-WG, to the candidate endogenous AHR ligand 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct that exhibits high potency in mammalian systems. FICZ readily induced mRNAs for CYP1A6 and CYP1A7. Cells exposed to FICZ for 3 hours expressed up to 5-fold greater quantities of CYP1A6/7 mRNAs than those exposed for 24 hours, suggesting FICZ is metabolized following rapid enzyme induction. FICZ appeared more potent than TCDD. Following a 3-hr exposure, the EC 50 for CYP1A6 mRNA induction by FICZ was ~6 nM, while the TCDD response was greater than 174 nM. These potencies were lower than those determined for mouse hepatoma cells (Hepa1c1c7; EC 50 = ~0.06 nM each). The difference in ligand potency between cell lines was confirmed by induction of ethoxyresorufin-O-deethylase (EROD) activity. mRNA from XLK-WG cells treated with 100 nM FICZ, 100 nM TCDD, or vehicle was also analyzed on expression microarrays. FICZ altered the expression of 105 more transcripts than TCDD, and common targets were altered more dramatically by FICZ. Overall, these studies demonstrate that although FICZ is a less potent CYP1A inducer in frog cells than in mouse cells, the reduction is much less than for TCDD. Relative conservation of the FICZ response in a TCDD-insensitive species suggests its physiological importance as an AHR ligand.

Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the FrogXenopus Laevis: Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type

“…High TCDD concentrations in this and subsequent experiments were necessary due to the relatively low TCDD binding affinity of AHRs from X. laevis and other amphibians (Laub et al, 2010;Lavine et al, 2005;Shoots et al, 2015). Neither AHR1a-A3 nor AHR1b-B1 was able to induce XRE-driven luciferase expression from the pGudLuc6.1 reporter plasmid (Figs.…”

“…XLK-WG cells (ATCC, Manassas, Virginia; (Martin et al, 1998) were cultured as directed by the supplier: 29 C with 5% CO 2 in RPMI-1640 medium (ThermoFisher Scientific, Waltham, MA) with 20% FBS (Invitrogen, Carlsbad, California; Iwamoto et al, 2012;Laub et al, 2010).…”

“…Fast growing and easily cared for, XLK-WG cells represent an excellent opportunity to employ the power of a frog tissue culture model to investigate subfunctionalization of the X. laevis AHR paralogs. We have previously pursued their use, publishing characterizations of the expression and function of AHRs along with the genes (Laub et al, 2010) and CYP1 enzyme activities they regulate (Iwamoto et al, 2012). Further, recent studies in AHR-deficient rats showed several defects related to the development of the kidney (Harrill et al, 2013), highlighting the potential importance of AHR in this tissue type and the relevance of AHR characterization in kidney-derived cells like XLK-WG.…”

“…The AHR also plays important roles in a wide range of vertebrate developmental and physiological processes (Gasiewicz and Henry, 2012), presumably through the action of endogenous ligands (Denison et al, 2011). 6-formylindolo [3,2-b]carbazole (FICZ), produced from tryptophan as a photoproduct, a result of oxidizing conditions, or as a metabolic product (Smirnova, 2016), is a wellcharacterized candidate endogenous agonist that exhibits even higher affinity for AHR than does TCDD (Laub et al, 2010;Wincent et al, 2009). The most prominent result of AHR activation is induced expression of the Cytochrome P4501A (CYP1A) subfamily of phase I detoxification enzymes (Hankinson, 1995), but numerous other transcripts are also altered directly or indirectly (eg, Frueh et al, 2001;Puga et al, 2000).…”

“…However, some gene duplicates show evidence of functional divergence (Chain and Evans, 2006;Hellsten et al, 2007). The objective of this study was to test the hypothesis that endogenous AHR1a and AHR1b have different molecular functions in XLK-WG (Martin et al, 1998), a frog cell line used previously for AHR characterization (Iwamoto et al, 2012;Laub et al, 2010). Using TALENs, we generated mutant cells lacking either AHR1a or AHR1b, examining the responsiveness of each to a xenobiotic and a candidate endogenous agonist.…”