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Archives of Toxicology

, Volume 92, Issue 12, pp 3471–3486 | Cite as

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

  • Constanze Knebel
  • Juliane Kebben
  • Ivano Eberini
  • Luca Palazzolo
  • Helen S. Hammer
  • Roderich D. Süssmuth
  • Tanja Heise
  • Stefanie Hessel-Pras
  • Alfonso Lampen
  • Albert Braeuning
  • Philip Marx-Stoelting
Molecular Toxicology
  • 171 Downloads

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.

Keywords

Triazole fungicides Nuclear receptor Aryl hydrocarbon receptor Liver toxicity Mixture toxicity Concentration additivity 

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

Notes

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.

Supplementary material

204_2018_2321_MOESM1_ESM.docx (228 kb)
Supplementary material 1 (DOCX 227 KB)
204_2018_2321_MOESM2_ESM.docx (23 kb)
Supplementary material 2 (DOCX 22 KB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Constanze Knebel
    • 1
  • Juliane Kebben
    • 1
  • Ivano Eberini
    • 2
  • Luca Palazzolo
    • 2
  • Helen S. Hammer
    • 3
  • Roderich D. Süssmuth
    • 4
  • Tanja Heise
    • 5
  • Stefanie Hessel-Pras
    • 1
  • Alfonso Lampen
    • 1
  • Albert Braeuning
    • 1
  • Philip Marx-Stoelting
    • 6
  1. 1.Department of Food SafetyGerman Federal Institute for Risk AssessmentBerlinGermany
  2. 2.Dipartimento di Scienze Farmacologiche e BiomolecolariUniversità degli Studi di MilanoMilanItaly
  3. 3.Natural and Medical Sciences InstituteUniversity of TübingenReutlingenGermany
  4. 4.Institute of ChemistryTechnical University BerlinBerlinGermany
  5. 5.Department of Pesticides SafetyGerman Federal Institute for Risk AssessmentBerlinGermany
  6. 6.German Centre for the Protection of Laboratory Animals (Bf3R)German Federal Institute for Risk AssessmentBerlinGermany

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