Abstract
Consumers are exposed to pesticide residues and other food contaminants via the diet. Both 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 constitutive androstane receptor (CAR) or the pregnane X receptor (PXR). We now investigated the effects of Pi on the aryl hydrocarbon receptor (AHR) and possible mixture toxicity when Pi is present in combination with BbF, an AHR ligand. In silico docking simulations indicate that Pi can bind to human AHR. Subsequent dual luciferase reporter gene assays in human HepG2 cells showed that Pi activates the AHR in vitro. This concentration-dependent activation was confirmed by real-time RT-PCR analyses of the model AHR target genes CYP1A1 and CYP1A2 in human HepaRG and HepG2 cells. In addition, induction of CYP1A1 protein levels and enzyme activity were recorded. Similarly, increased mRNA expression and enzyme activity of Cyp1a1 and Cyp1a2 was observed in livers of rats treated with Pi for 28 days via the diet. 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. Finally, mixture effects of Pi and BbF were analyzed in human cell lines: modeling of concentration–response curves revealed concentration additivity. In conclusion, our results demonstrate that the triazole Pi is an activator of AHR in silico, in vitro and in vivo and causes additive effects with an established AHR ligand.
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Abbreviations
- AHR:
-
Aryl hydrocarbon receptor
- AOP:
-
Adverse outcome pathway
- BaP:
-
Benzo[a]pyrene
- BbF:
-
Benzo[b]fluoranthene
- CAR:
-
Constitutive androstane receptor
- CITCO:
-
6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4dichlorobenzyl)oxime
- CYP:
-
Cytochrome P450
- DMSO:
-
Dimethylsulfoxide
- EFSA:
-
European Food Safety Authority
- EROD:
-
Ethoxyresorufin-O-deethylase
- FCS:
-
Fetal calf serum
- HIF-2α:
-
Hypoxia-inducible factor 2α
- IARC:
-
International Agency for the Research on Cancer
- IPA:
-
Ingenuity pathway analysis
- LBD:
-
Ligand binding domain
- LLOQ:
-
Lower limits of quantification
- MTT:
-
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- PAH:
-
Polycyclic aromatic hydrocarbon
- Pi:
-
Propiconazole
- PPAR:
-
Peroxisome proliferator-induced receptor
- PXR:
-
Pregnane X receptor
- RSLC:
-
Rapid separation LC
- SD:
-
Standard deviation
- TCDD:
-
2,3,7,8-Tetrachlorodibenzo[p]dioxin
- TCPOBOP:
-
1,4-Bis-[2-(3,5-dichloropyridyloxy)] benzene
- tSIM:
-
Targeted single-ion monitoring
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Acknowledgements
The authors thank Jannika Neeb and Inês Aragão for technical assistance as well as Dr. Claudia Luckert, Dr. Josef Rasinger and Dr. Axel Oberemm for helpful guidance with data analysis. This work was supported by the German Federal Institute for Risk Assessment (Grant 1322-499 and 657, PMS and AB) and by Grants from Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR)-“Progetto Eccellenza” (IE). IE gratefully acknowledges departmental “Linea 2-Azione A 2017” funding.
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Knebel, C., Kebben, J., Eberini, I. et al. Propiconazole is an activator of AHR and causes concentration additive effects with an established AHR ligand. Arch Toxicol 92, 3471–3486 (2018). https://doi.org/10.1007/s00204-018-2321-x
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DOI: https://doi.org/10.1007/s00204-018-2321-x