Elsevier

Food and Chemical Toxicology

Volume 123, January 2019, Pages 481-491
Food and Chemical Toxicology

The azole fungicide tebuconazole affects human CYP1A1 and CYP1A2 expression by an aryl hydrocarbon receptor-dependent pathway

https://doi.org/10.1016/j.fct.2018.11.039Get rights and content

Highlights

  • Tebuconazole activates transcription of AHR-dependent genes in rat liver in vivo.

  • Tebuconazole activates transcription of AHR-dependent genes in human cell lines.

  • Additive effects are observed with tebuconazole/propiconazole mixtures.

Abstract

Tebuconazole, a member of the triazole group of fungicides, exerts hepatotoxicity in rodent studies. Knowledge on the molecular mechanisms underlying tebuconazole toxicity is limited. Previous studies suggest that activation of xenobiotic-sensing nuclear receptors plays a role in triazole fungicide-mediated hepatotoxicity. This study aimed to characterize the ability of tebuconazole to activate gene expression via the aryl hydrocarbon receptor (AHR). Results demonstrate a statistically significant induction of the AHR target genes CYP1A1 and CYP1A2 in HepG2 and HepaRG human liver cells in vitro at concentrations corresponding to tebuconazole tissue levels reached under subtoxic conditions in vivo. CYP1A1 and CYP1A2 induction was abolished in the presence of an AHR antagonist or in AHR-knockout HepaRG cells, substantiating the importance of the AHR for the observed effects. Although the results indicate that tebuconazole is a weak inducer of AHR-dependent genes, combined exposure of HepaRG cells to tebuconazole and the previously identified AHR agonist propiconazole showed additive effects on CYP1A1 and CYP1A2 expression. In summary, we demonstrate that AHR-downstream gene expression is affected by tebuconazole in an AHR-dependent manner. Data indicate that dose addition may be assumed for the assessment of AHR-related effects of triazole fungicide mixtures.

Introduction

Triazole fungicides are used in agriculture and in human and veterinary medicine to treat a broad spectrum of fungal disease. They are designed to inhibit fungal cytochrome P450 (CYP) 51 (lanosterol-14α-demethylase) to block cell membrane synthesis (Georgopapadakou, 1998). As a side effect they may inhibit mammalian CYP enzymes in an unspecific manner, among them are also some CYPs essential for synthesis of steroid hormones (e.g. aromatase, CYP19A1) or for the metabolism of vitamins such as vitamin A (Robinson et al., 2012; Vinggaard et al., 2006; Zarn et al., 2003). It is therefore not surprising that triazole fungicides may cause adverse effects related to this mode of action. In studies conducted for the regulatory approval of tebuconazole as an active substance for pesticidal use, for example, the compound caused effects in steroidogenic endocrine organs and developmental toxicity. Tebuconazole induced hypertrophy of adrenal glands in oral short-term toxicity studies in rats and dogs, and was also classified for developmental toxicity (H361d) due to malformations observed in several species (EFSA, 2014).

In general, however, the most sensitive target organ of triazoles is the liver. This is, at least in part, attributed to the fact that these substances also interact with hepatic nuclear receptors such as the constitutive androstane receptor (CAR) (Goetz and Dix, 2009). Hence it is not surprising that recently tebuconazole was also shown to cause effects on the liver in vivo (EFSA, 2014; Schmidt et al., 2016). In short term studies tebuconazole exposure led to liver weight increase and centrilobular hypertrophy in rats and mice (EFSA, 2014; Schmidt et al., 2016). In mice but not in rats tebuconazole additionally caused hepatocellular tumors in long-term studies, which was not considered relevant for classification due to assumed species differences between mice and men (EFSA, 2014).

Indeed, triazole-induced liver effects may exhibit significant species differences. For example, Marx-Stoelting et al. (2017) showed that hepatic responses to another azole fungicide (cyproconazole) were remarkably less pronounced in mice with humanized CAR and pregnane-X-receptor (PXR) as compared to wild-type controls, suggesting that the compound was not able to stimulate the receptors from the two species to a comparable degree. Species differences between rodents and humans with regard to the hepatic consequences of CAR activation, especially tumor findings in animal studies, are controversially discussed among toxicologists (Braeuning, 2014; Braeuning et al., 2015; Braeuning and Schwarz, 2016; Elcombe et al., 2014; Yamada et al., 2015).

Tamura et al. (2013, 2015) suggested that activation of CAR may not be responsible for tebuconazole-dependent liver effects observed in mice. Furthermore, in a recent study it was shown that tebuconazole is not an agonist but an antagonist of human and rat CAR (Knebel et al., 2018b). Hence, tebuconazole-mediated toxicity most likely cannot be attributed to activation of CAR alone, but other hepatic nuclear receptors have to be considered. Indeed, data from global gene expression analysis of some azole fungicides suggest the involvement of other nuclear receptors than just CAR in azole-mediated liver toxicity (e.g. see Goetz and Dix (2009) and Heise et al. (2015)). Among them are PXR (activation by tebuconazole already analyzed by Knebel et al. (2018b)) and the aryl hydrocarbon receptor (AHR). Since other structurally related substances like prochloraz or propiconazole have already been shown to activate AHR (Halwachs et al., 2013; Knebel et al., 2018a; Rieke et al., 2014) and tebuconazole has been shown to activate CYP1A1 and Cyp1a1/2 activity in EROD/MROD (7-ethoxy/methoxyresorufin-O deethylase) assays (Sergent et al., 2009; Yang et al., 2018) it is reasonable to test the potential of tebuconazole to activate human and murine AHR.

In this study we therefore investigated the potential of tebuconazole to activate the AHR in vivo and in vitro, in rat liver and in human cell lines of hepatic origin.

Section snippets

Test compounds

Technical grade tebuconazole (CAS no. 107534-96-3; Batch no. NK21BX0392; purity 96.20%) as applied in plant protection products was obtained from Bayer (Leverkusen, Germany). Analytical grade tebuconazole (CAS no. 107534-96-3; Batch no. G142375; purity 99.30%) was purchased from LGC Standards (Wesel, Germany). The AHR inhibitor CH-223191 (CAS no. 301326-22-7; Batch no. 12131228) was purchased from Sigma-Aldrich (Taufkirchen, Germany) and the AHR agonist 3-methylcholanthrene (CAS no. 56-49-5;

Tebuconazole induces Cyp1a1 and Cyp1a2 in rats in vivo

In the course of the evaluation of a previous in vivo study in rats exposed to up to 1000 ppm (corresponding to a mean daily intake of 71.24 (±4.38) mg/kg bw) tebuconazole via the diet for 28 days, real-time RT-PCR analyses revealed that the highest dose of tebuconazole caused a statistically significant induction of the expression of the mRNAs encoded by the Cyp1a1 and, to a lesser extent, Cyp1a2 genes (Fig. 1A and B). Accordingly, enzymatic activity of Cyp1a1 was significantly up-regulated in

Discussion

The present data demonstrate that tebuconazole is capable of activating AHR-dependent genes not only in rodent, but also in human liver cells. Data further indicate that tebuconazole is a weak activator of these genes, as compared to model AHR ligands and also in comparison with the structurally related fungicide propiconazole (Knebel et al., 2018a). The low micromolar concentrations at which first effects of tebuconazole on AHR-dependent genes were observed in vitro are similar to the

Acknowledgment

This study was supported by the German Federal Institute for Risk Assessment [grant 1322-657 and 1322-499] and by the Robert Bosch Foundation, Stuttgart, Germany. We wish to thank Dr. Lars Niemann for making available tissue samples from animal studies. Furthermore, we thank Dr. Claudia Luckert and Dr. Axel Oberemm for helpful guidance with the analysis.

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    PMS and AB contributed equally to this paper.

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