Abstract
Triazoles are commonly used fungicides which show liver toxicity in rodent studies. While hepatocellular hypertrophy is the most prominent finding, some triazoles have also been reported to cause hepatocellular steatosis. The aim of our study was to elucidate molecular mechanisms of triazole-mediated steatosis. Therefore, we used the two triazoles propiconazole (Pi) and tebuconazole (Te) as test compounds in in vitro assays using the human hepatocarcinoma cell lines HepG2 and HepaRG. Triglyceride accumulation was measured using the Adipored assay and by a gas-chromatographic method. Reporter gene analyses were used to assess the ability of Pi and Te to activate nuclear receptors, which are described as the molecular initiators in the adverse outcome pathway (AOP) for liver steatosis. The expression of steatosis-associated genes was investigated by RT-PCR. Mechanistic analyses of triazole-mediated steatosis were performed using HepaRG subclones that are deficient in different nuclear receptors. Pi and Te both interacted with the constitutive androstane receptor (CAR), the peroxisome proliferator-activated receptor alpha (PPARα), and the pregnane X receptor (PXR). Both compounds induced expression of steatosis-related genes and cellular triglyceride accumulation. The knockout of PXR in HepaRG cells, but not the CAR knockout, abolished triazole-induced triglyceride accumulation, thus underlining the crucial role of PXR in hepatic steatosis resulting from exposure to these fungicides. In conclusion, our findings provide new insight into the molecular mechanisms of steatosis induction by triazole fungicides and identify PXR as a critical mediator of this process.
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References
Al-Eryani L, Wahlang B, Falkner KC et al (2015) Identification of environmental chemicals associated with the development of toxicant-associated fatty liver disease in rodents. Toxicol Pathol 43(4):482–497. https://doi.org/10.1177/0192623314549960
Angrish MM, Kaiser JP, McQueen CA, Chorley BN (2016) Tipping the balance: hepatotoxicity and the 4 apical key events of hepatic steatosis. Toxicol Sci 150(2):261–268. https://doi.org/10.1093/toxsci/kfw018
Ankley GT, Edwards SW (2018) The Adverse outcome pathway: a multifaceted framework supporting 21(st) century toxicology. Curr Opin Toxicol 9:1–7. https://doi.org/10.1016/j.cotox.2018.03.004
Bitter A, Rummele P, Klein K et al (2015) Pregnane X receptor activation and silencing promote steatosis of human hepatic cells by distinct lipogenic mechanisms. Arch Toxicol 89(11):2089–2103. https://doi.org/10.1007/s00204-014-1348-x
Braeuning A, Sanna R, Huelsken J, Schwarz M (2009) Inducibility of drug-metabolizing enzymes by xenobiotics in mice with liver-specific knockout of Ctnnb1. Drug Metab Dispos 37(5):1138–1145. https://doi.org/10.1124/dmd.108.026179
Braeuning A, Vetter S, Orsetti S, Schwarz M (2012) Paradoxical cytotoxicity of tert-butylhydroquinone in vitro: what kills the untreated cells? Arch Toxicol 86(9):1481–1487. https://doi.org/10.1007/s00204-012-0841-3
Dentin R, Girard J, Postic C (2005) Carbohydrate responsive element binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c): two key regulators of glucose metabolism and lipid synthesis in liver. Biochimie 87(1):81–86. https://doi.org/10.1016/j.biochi.2004.11.008
Donato MT, Tolosa L, Jimenez N, Castell JV, Gomez-Lechon MJ (2012) High-content imaging technology for the evaluation of drug-induced steatosis using a multiparametric cell-based assay. J Biomol Screen 17(3):394–400. https://doi.org/10.1177/1087057111427586
EFSA (2015) The 2013 European Union report on pesticide residues in food. EFSA J 13(3):4038
EFSA (2012) Identification of cumulative assessment groups of pesticides, vol 9. EFSA Supporting Publications, Parma, Italy, pp 1-303
Georgopapadakou NH (1998) Antifungals: mechanism of action and resistance, established and novel drugs. Curr Opin Microbiol 1(5):547–557
Goetz AK, Dix DJ (2009) Mode of action for reproductive and hepatic toxicity inferred from a genomic study of triazole antifungals. Toxicol Sci 110(2):449–462. https://doi.org/10.1093/toxsci/kfp098
Gripon P, Rumin S, Urban S et al (2002) Infection of a human hepatoma cell line by hepatitis B virus. Proc Natl Acad Sci USA 99(24):15655–15660. https://doi.org/10.1073/pnas.232137699
Hampf M, Gossen M (2006) A protocol for combined Photinus and Renilla luciferase quantification compatible with protein assays. Anal Biochem 356(1):94–99. https://doi.org/10.1016/j.ab.2006.04.046
Heise T, Schmidt F, Knebel C et al (2015) Hepatotoxic effects of (tri)azole fungicides in a broad dose range. Arch Toxicol 89(11):2105–2117. https://doi.org/10.1007/s00204-014-1336-1
Heise T, Schmidt F, Knebel C et al (2018) Hepatotoxic combination effects of three azole fungicides in a broad dose range. Arch Toxicol 92(2):859–872. https://doi.org/10.1007/s00204-017-2087-6
Hester SD, Nesnow S (2008) Transcriptional responses in thyroid tissues from rats treated with a tumorigenic and a non-tumorigenic triazole conazole fungicide. Toxicol Appl Pharmacol 227(3):357–369. https://doi.org/10.1016/j.taap.2007.10.030
Joshi-Barve S, Kirpich I, Cave MC, Marsano LS, McClain CJ (2015) Alcoholic, nonalcoholic, and toxicant-associated steatohepatitis: mechanistic similarities and differences. Cell Mol Gastroenterol Hepatol 1(4):356–367. https://doi.org/10.1016/j.jcmgh.2015.05.006
Kienhuis AS, Slob W, Gremmer ER, Vermeulen JP, Ezendam J (2015) A Dose-response modeling approach shows that effects from mixture exposure to the skin sensitizers isoeugenol and cinnamal are in line with dose addition and not with synergism. Toxicol Sci 147(1):68–74. https://doi.org/10.1093/toxsci/kfv109
Kleiner DE, Brunt EM, Van Natta M et al (2005) Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 41(6):1313–1321. https://doi.org/10.1002/hep.20701
Knebel C, Kebben J, Eberini I et al (2018a) Propiconazole is an activator of AHR and causes concentration additive effects with an established AHR ligand. Arch Toxicol 92:3471–3486. https://doi.org/10.1007/s00204-018-2321-x
Knebel C, Neeb J, Zahn E et al (2018b) Unexpected effects of propiconazole, tebuconazole, and their mixture on the receptors CAR and PXR in human liver cells. Toxicol Sci 163(1):170–181. https://doi.org/10.1093/toxsci/kfy026
Knebel C, Heise T, Zanger UM, Lampen A, Marx-Stoelting P, Braeuning A (2019) The azole fungicide tebuconazole affects human CYP1A1 and CYP1A2 expression by an aryl hydrocarbon receptor-dependent pathway. Food Chem Toxicol 123:481–491. https://doi.org/10.1016/j.fct.2018.11.039
Kortenkamp A, Backhaus T, Faust M (2009) State of the art report on mixture toxicity. EU Report. European Commission, Brussels, Belgium
Luckert C, Braeuning A, de Sousa G et al (2018) Adverse outcome pathway-driven analysis of liver steatosis in vitro: a case study with cyproconazole. Chem Res Toxicol 31(8):784–798. https://doi.org/10.1021/acs.chemrestox.8b00112
Marx-Stoelting P, Ganzenberg K, Knebel C et al (2017) Hepatotoxic effects of cyproconazole and prochloraz in wild-type and hCAR/hPXR mice. Arch Toxicol 91:2895–2907. https://doi.org/10.1007/s00204-016-1925-2
Mellor CL, Steinmetz FP, Cronin MT (2015) The identification of nuclear receptors associated with hepatic steatosis to develop and extend adverse outcome pathways. Crit Rev Toxicol 46:1–15. https://doi.org/10.3109/10408444.2015.1089471
Noureddin M, Rinella ME (2015) Nonalcoholic fatty liver disease, diabetes, obesity, and hepatocellular carcinoma. Clin Liver Dis 19(2):361–379. https://doi.org/10.1016/j.cld.2015.01.012
Prutner W, Nicken P, Haunhorst E, Hamscher G, Steinberg P (2013) Effects of single pesticides and binary pesticide mixtures on estrone production in H295R cells. Arch Toxicol 87(12):2201–2214. https://doi.org/10.1007/s00204-013-1081-x
Rieke S, Heise T, Schmidt F et al (2017) Mixture effects of azole fungicides on the adrenal gland in a broad dose range. Toxicology 385:28–37. https://doi.org/10.1016/j.tox.2017.04.012
Roth A, Looser R, Kaufmann M et al (2008) Regulatory cross-talk between drug metabolism and lipid homeostasis: constitutive androstane receptor and pregnane X receptor increase Insig-1 expression. Mol Pharmacol 73(4):1282–1289. https://doi.org/10.1124/mol.107.041012
Sae-Lee C, Moolsuwan K, Chan L, Poungvarin N (2016) ChREBP regulates itself and metabolic genes implicated in lipid accumulation in beta-cell line. PLoS One 11(1):e0147411. https://doi.org/10.1371/journal.pone.0147411
Schmidt F, Marx-Stoelting P, Haider W et al (2016) Combination effects of azole fungicides in male rats in a broad dose range. Toxicology 355–356:54–63. https://doi.org/10.1016/j.tox.2016.05.018
Shimano H (2001) Sterol regulatory element-binding proteins (SREBPs): transcriptional regulators of lipid synthetic genes. Prog Lipid Res 40(6):439–452
Spruiell K, Jones DZ, Cullen JM, Awumey EM, Gonzalez FJ, Gyamfi MA (2014) Role of human pregnane X receptor in high fat diet-induced obesity in pre-menopausal female mice. Biochem Pharmacol 89(3):399–412. https://doi.org/10.1016/j.bcp.2014.03.019
Spruiell K, Richardson RM, Cullen JM, Awumey EM, Gonzalez FJ, Gyamfi MA (2014) Role of pregnane X receptor in obesity and glucose homeostasis in male mice. J Biol Chem 289(6):3244–3261. https://doi.org/10.1074/jbc.M113.494575
Stepankova M, Pastorkova B, Bachleda P, Dvorak Z (2017) Itraconazole cis-diastereoisomers activate aryl hydrocarbon receptor AhR and pregnane X receptor PXR and induce CYP1A1 in human cell lines and human hepatocytes. Toxicology 383:40–49. https://doi.org/10.1016/j.tox.2017.04.002
Tanner N, Kubik L, Luckert C et al (2018) Regulation of drug metabolism by the interplay of inflammatory signaling, steatosis, and xeno-sensing receptors in HepaRG cells. Drug Metab Dispos 46(4):326–335. https://doi.org/10.1124/dmd.117.078675
Tolosa L, Gomez-Lechon MJ, Jimenez N, Hervas D, Jover R, Donato MT (2016) Advantageous use of HepaRG cells for the screening and mechanistic study of drug-induced steatosis. Toxicol Appl Pharmacol 302:1–9. https://doi.org/10.1016/j.taap.2016.04.007
Tonazzi A, Eberini I, Indiveri C (2013) Molecular mechanism of inhibition of the mitochondrial carnitine/acylcarnitine transporter by omeprazole revealed by proteoliposome assay, mutagenesis and bioinformatics. PLoS One 8(12):e82286. https://doi.org/10.1371/journal.pone.0082286
Tully DB, Bao W, Goetz AK et al (2006) Gene expression profiling in liver and testis of rats to characterize the toxicity of triazole fungicides. Toxicol Appl Pharmacol 215(3):260–273. https://doi.org/10.1016/j.taap.2006.02.015
Vinken M (2013) The adverse outcome pathway concept: a pragmatic tool in toxicology. Toxicology 312:158–165. https://doi.org/10.1016/j.tox.2013.08.011
Vinken M (2015) Adverse outcome pathways and drug-induced liver injury testing. Chem Res Toxicol 28(7):1391–1397. https://doi.org/10.1021/acs.chemrestox.5b00208
Vinken M (2016) Adverse outcome pathways as tools to assess drug-induced toxicity. Methods Mol Biol 1425:325–337. https://doi.org/10.1007/978-1-4939-3609-0_14 (Clifton, NJ)
White DL, Kanwal F, El-Serag HB (2012) Association between nonalcoholic fatty liver disease and risk for hepatocellular cancer, based on systematic review. Clin Gastroenterol Hepatol 10(12):1342–1359.e2. https://doi.org/10.1016/j.cgh.2012.10.001
Zahn E, Wolfrum J, Knebel C et al (2018) Mixture effects of two plant protection products in liver cell lines. Food Chem Toxicol 112:299–309. https://doi.org/10.1016/j.fct.2017.12.067
Acknowledgements
The authors thank Regina Al-Hamwi, Marlies Sagmeister, Jaqueline Just, and Inês Aragão for technical assistance. Furthermore, we thank Dr. Claudia Luckert and Dr. Axel Oberemm for helpful guidance with the analysis. This work was supported by the German Federal Institute for Risk Assessment (Grants 1322-657 and 1322-499).
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Knebel, C., Buhrke, T., Süssmuth, R. et al. Pregnane X receptor mediates steatotic effects of propiconazole and tebuconazole in human liver cell lines. Arch Toxicol 93, 1311–1322 (2019). https://doi.org/10.1007/s00204-019-02445-2
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DOI: https://doi.org/10.1007/s00204-019-02445-2