Unexpected Effects of Propiconazole, Tebuconazole, and Their Mixture on the Receptors CAR and PXR in Human Liver Cells

Knebel, Constanze; Neeb, Jannika; Zahn, Elisabeth; Schmidt, Folke; Carazo, Alejandro; Holas, Ondřej; Pávek, Petr; Püschel, Gerhard; Zanger, Ulrich M.; Lampen, Alfonso; Marx‐Stoelting, Philip; Braeuning, Albert

doi:10.1093/toxsci/kfy026

Cited by 33 publications

(52 citation statements)

References 38 publications

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“…Recently published data on CAR activation by other triazole fungicides confirm the functionality of the reporter assay51 , thereby demonstrating the potent murine CAR agonist cyproconazole does not activate human CAR.The present AOP 60 ("NR1I2 (Pregnane X Receptor, PXR) activation leading to hepatic steatosis") at www.aopwiki.org and the proposed AOP network by Mellor et al 12 depict PXR activation as a molecular initiating event followed by the key event CD36 upregulation. This designation is in line with literature reports of PXR-dependent CD36 induction52,53 and associated with an increase in cellular fatty acid influx from external sources according to the AOP.…”

supporting

confidence: 65%

Adverse Outcome Pathway-Driven Analysis of Liver Steatosis in Vitro: A Case Study with Cyproconazole

Luckert

Braeuning

Sousa³

et al. 2018

Chem. Res. Toxicol.

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Adverse outcome pathways (AOPs) describe causal relationships between molecular perturbation and adverse cellular effects and are being increasingly adopted for linking in vitro mechanistic toxicology to in vivo data from regulatory toxicity studies. In this work, a case study was performed by developing a bioassay toolbox to assess key events in the recently proposed AOP for chemically induced liver steatosis. The toolbox is comprised of in vitro assays to measure nuclear receptor activation, gene and protein expression, lipid accumulation, mitochondrial respiration, and formation of fatty liver cells. Assay evaluation was performed in human HepaRG hepatocarcinoma cells exposed to the model compound cyproconazole, a fungicide inducing steatosis in rodents. Cyproconazole dose-dependently activated RARα and PXR, two molecular initiating events in the steatosis AOP. Moreover, cyproconazole provoked a disruption of mitochondrial functions and induced triglyceride accumulation and the formation of fatty liver cells as described in the AOP. Gene and protein expression analysis, however, showed expression changes different from those proposed in the AOP, thus suggesting that the current version of the AOP might not fully reflect the complex mechanisms linking nuclear receptor activation and liver steatosis. Our study shows that cyproconazole induces steatosis in human liver cells in vitro and demonstrates the utility of systems-based approaches in the mechanistic assessment of molecular and cellular key events in an AOP. AOP-driven in vitro testing as demonstrated can further improve existing AOPs, provide insight regarding molecular mechanisms of toxicity, and inform predictive risk assessment.

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supporting

confidence: 65%

Adverse Outcome Pathway-Driven Analysis of Liver Steatosis in Vitro: A Case Study with Cyproconazole

Luckert

Braeuning

Sousa³

et al. 2018

Chem. Res. Toxicol.

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“…Reporter gene assays and mRNA induction studies show that propiconazole is a moderate activator of human and rodent CAR. This is supported by mouse and rat studies in vivo [ 56 , 174 ]. At the same time, propiconazole activates PXR in all three species [ 55 ].…”

Section: Metabolic Effects Of Edc Classes Potentially Mediated By mentioning

confidence: 63%

Metabolism-Disrupting Chemicals and the Constitutive Androstane Receptor CAR

Küblbeck

Niskanen

Honkakoski

2020

Cells

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During the last two decades, the constitutive androstane receptor (CAR; NR1I3) has emerged as a master activator of drug- and xenobiotic-metabolizing enzymes and transporters that govern the clearance of both exogenous and endogenous small molecules. Recent studies indicate that CAR participates, together with other nuclear receptors (NRs) and transcription factors, in regulation of hepatic glucose and lipid metabolism, hepatocyte communication, proliferation and toxicity, and liver tumor development in rodents. Endocrine-disrupting chemicals (EDCs) constitute a wide range of persistent organic compounds that have been associated with aberrations of hormone-dependent physiological processes. Their adverse health effects include metabolic alterations such as diabetes, obesity, and fatty liver disease in animal models and humans exposed to EDCs. As numerous xenobiotics can activate CAR, its role in EDC-elicited adverse metabolic effects has gained much interest. Here, we review the key features and mechanisms of CAR as a xenobiotic-sensing receptor, species differences and selectivity of CAR ligands, contribution of CAR to regulation hepatic metabolism, and evidence for CAR-dependent EDC action therein.

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“…We couldn't observe any AHR activation after treatment with cyproconazole or tebuconazole when tested with the sensitive endpoint of luciferase reporter analysis up to the highest possible non-cytotoxic concentrations (own unpublished data). Instead, cyproconazole acts as an activator of human and rodent PXR and also rodent but not human CAR (Marx-Stoelting et al, 2017), whereas tebuconazole also activates PXR but is an antagonist of CAR (Knebel et al, 2018). Together these observations demonstrate that despite evident similarities at the level of chemical structure, the molecular targets of these substances differ considerably.…”

Section: Discussionmentioning

confidence: 92%

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

et al. 2018

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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.

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Unexpected Effects of Propiconazole, Tebuconazole, and Their Mixture on the Receptors CAR and PXR in Human Liver Cells

Cited by 33 publications

References 38 publications

Adverse Outcome Pathway-Driven Analysis of Liver Steatosis in Vitro: A Case Study with Cyproconazole

Adverse Outcome Pathway-Driven Analysis of Liver Steatosis in Vitro: A Case Study with Cyproconazole

Metabolism-Disrupting Chemicals and the Constitutive Androstane Receptor CAR

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

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