The azole fungicide tebuconazole affects human CYP1A1 and CYP1A2 expression by an aryl hydrocarbon receptor-dependent pathway
Graphical abstract
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.
References (31)
Antifungals: mechanism of action and resistance, established and novel drugs
Curr. Opin. Microbiol.
(1998)- et al.
Effects of currently used pesticides and their mixtures on the function of thyroid hormone and aryl hydrocarbon receptor in cell culture
Toxicol. Appl. Pharmacol.
(2015) - et al.
Triazole induced concentration-related gene signatures in rat whole embryo culture
Reprod. Toxicol.
(2012) - et al.
Combination effects of azole fungicides in male rats in a broad dose range
Toxicology
(2016) - et al.
CYP1A1 induction and CYP3A4 inhibition by the fungicide imazalil in the human intestinal Caco-2 cells-comparison with other conazole pesticides
Toxicol. Lett.
(2009) - et al.
Involvement of constitutive androstane receptor in liver hypertrophy and liver tumor development induced by triazole fungicides
Food Chem. Toxicol.
(2015) - et al.
Dose-response involvement of constitutive androstane receptor in mouse liver hypertrophy induced by triazole fungicides
Toxicol. Lett.
(2013) - et al.
An introduction to the molecular basics of aryl hydrocarbon receptor biology
Biol. Chem.
(2010) Liver cell proliferation and tumor promotion by phenobarbital: relevance for humans?
Arch. Toxicol.
(2014)- et al.
Model systems for understanding mechanisms of nongenotoxic carcinogenesis: response
Toxicol. Sci.
(2015)
Is the question of phenobarbital as potential liver cancer risk factor for humans really resolved?
Arch. Toxicol.
Paradoxical cytotoxicity of tert-butylhydroquinone in vitro: what kills the untreated cells?
Arch. Toxicol.
Conclusion on the peer review of the pesticide risk assessment of the active substance tebuconazole
EFSA J.
Mode of action and human relevance analysis for nuclear receptor-mediated liver toxicity: a case study with phenobarbital as a model constitutive androstane receptor (CAR) activator
Crit. Rev. Toxicol.
Profiling induction of cytochrome p450 enzyme activity by statins using a new liquid chromatography-tandem mass spectrometry cocktail assay in human hepatocytes
Drug Metabol. Dispos.
Cited by (32)
A comprehensive review on toxicological mechanisms and transformation products of tebuconazole: Insights on pesticide management
2024, Science of the Total EnvironmentA novel Ti-based magnetic MOFs combined with COF for tetracycline and tebuconazole removal from water
2023, Journal of Environmental Chemical EngineeringThe fungicide tebuconazole modulates the sodium current of human Na<inf>V</inf>1.5 channels expressed in HEK293 cells
2023, Food and Chemical ToxicologyDetrimental consequences of tebuconazole on redox homeostasis and fatty acid profile of honeybee brain
2023, Insect Biochemistry and Molecular BiologyElectrochemical behavior of fungicide tebuconazole and its voltammetric determination on an oxygen-terminated boron-doped diamond electrode
2023, Journal of Electroanalytical Chemistry
- 1
PMS and AB contributed equally to this paper.