Propiconazole is an activator of AHR and causes concentration additive effects with an established AHR ligand

Abstract

Pesticides can exert adverse effects on different target organs via the activation of nuclear receptor pathways. Hepatotoxic effects of the widely used triazole fungicide propiconazole (Pi) are generally attributed to the activation of the ligand-activated transcription factor constitutive androstane receptor (CAR) or the pregnane X receptor (PXR). We now investigated the effects of Pi on the ligand-activated transcription factor aryl hydrocarbon receptor AHR. Therefore, the effects of Pi on AHR-dependent reporter gene transactivation, AHR target gene mRNA and protein expression as well as enzyme activities were examined in HepG2 or HepaRG human hepatoma cells using dual luciferase assays, RT-PCR, mass spectrometry or the ethoxyresorufin-O-deethylase assay, respectively. Additionally mRNA expression and enzyme activity was analyzed in rat liver ex vivo. Furthermore, AHR target gene expression was measured in AHR-, CAR-, and PXR-knockout HepaRG cells. Subsequently, we investigated combination effects of Pi and the model AHR ligand benzo[b]fluoranthene (BbF) in vitro. Our results show that Pi activates the AHR in reporter gene assays in human HepG2 cells. This dose-dependent activation of the AHR was subsequently confirmed by real-time RT-PCR analyses of the model AHR target genes CYP1A1 and CYP1A2 in human HepaRG cells and in livers of rats treated with Pi for 28 days via the diet. In addition, results of CYP1A1 protein level quantification showed Pi-dependent induction of the enzyme. CYP1A1 enzyme activity measurements demonstrate that higher concentrations of Pi significantly induce CYP1A1-dependent reactions in human HepaRG cells and in rat liver. Gene expression analysis in AHR-knockout HepaRG cells showed no induction of CYP1A1 and CYP1A2, whereas gene expression in CAR-, and PXR-knockout cells was induced. Furthermore, mixture effects of Pi and BbF were observed in human cell lines. Modeling of dose-response curves revealed that these cellular effects can be explained by concentration additivity. In conclusion, our results demonstrate that the triazole Pi is an activator of AHR.

The Connection of Azole Fungicides with Xeno-Sensing Nuclear Receptors, Drug Metabolism and Hepatotoxicity

“…For human liver cells, in vitro evidence at the reporter gene, target mRNA, protein, and enzyme activity level suggests weak agonism of propiconazole at the AHR, substantiated by in silico molecular docking study results [16,26,27,31,35,37,38]. Additionally, in vitro experiments using a human AHR antagonist, AHR binding site-mutated reporter variants or AHR knockout cells mechanistically strengthen the evidence for AHR activation by propiconazole [31]. Target mRNA [5] [31,37,38] Target protein [26,31] Target enzyme activity [31,35,37] In vivo studies Target mRNA [6] [4,31,36] Target enzyme activity [36] [31,36] Similarly, in vivo rat and mouse data at the target mRNA and enzyme activity levels indirectly connect propiconazole with the activation of CAR [5,6,20,33,36,39,40] (Table 2).…”

“…Especially by using specific constructs in reporter gene assays, in vitro data contribute the bulk of direct evidence for interaction of a certain azole fungicide with one of the nuclear receptors of interest. Computational approaches such as molecular docking or toxicogenomic pathway analyses have played a role in a minority of past projects to identify the nuclear receptor-interacting potential of azoles [6,[31][32][33]. As a rule, the vast majority of effects observed were receptor activation and subsequent target gene induction.…”

“…Available literature suggests that propiconazole is a weak activator of mouse, rat, and human AHR (Table 1). Data for mouse and rat are mainly derived from in vivo studies that yielded indirect evidence for AHR activation via target gene and enzyme activity regulation [4,6,31,36]. For human liver cells, in vitro evidence at the reporter gene, target mRNA, protein, and enzyme activity level suggests weak agonism of propiconazole at the AHR, substantiated by in silico molecular docking study results [16,26,27,31,35,37,38].…”

“…Data for mouse and rat are mainly derived from in vivo studies that yielded indirect evidence for AHR activation via target gene and enzyme activity regulation [4,6,31,36]. For human liver cells, in vitro evidence at the reporter gene, target mRNA, protein, and enzyme activity level suggests weak agonism of propiconazole at the AHR, substantiated by in silico molecular docking study results [16,26,27,31,35,37,38]. Additionally, in vitro experiments using a human AHR antagonist, AHR binding site-mutated reporter variants or AHR knockout cells mechanistically strengthen the evidence for AHR activation by propiconazole [31].…”

Targeting the Aryl Hydrocarbon Receptor (AhR): A Review of the In-Silico Screening Approaches to Identify AhR Modulators

“…These developed models provided a clear basis towards understanding the mechanism of ligand-dependent activation of AHR via its PAS B domain. In particular, the above mentioned molecular docking and mutagenesis analyses helped in identifying and confirming the binding pocket of TCDD and other AhR modulators [46,51,54,55].…”