Hepatotoxic effects of cyproconazole and prochloraz in wild-type and hCAR/hPXR mice

Abstract

The agricultural fungicides cyproconazole and prochloraz exhibit hepatotoxicity in rodent studies and are tumorigenic following chronic exposure. Both substances are suspected to act via a CAR (constitutive androstane receptor)/PXR (pregnane-X-receptor)-dependent mechanism. Human relevance of these findings is under debate. A 28-day toxicity study was conducted in mice with humanized CAR and PXR (hCAR/hPXR) with two dose levels (50 or 500 ppm) of both substances, using the model CAR activator phenobarbital as a reference. Results were compared to wild-type mice. A treatment-related increase in liver weights was observed for all three substances at least at the high-dose level. Changes in the expression of classic CAR/PXR target genes such as Cyp2b10 were induced by cyproconazole and phenobarbital in both genotypes, while prochloraz treatment resulted in gene expression changes indicative of additional aryl hydrocarbon receptor activation, e.g. by up-regulation of Cyp1a1 expression. Cyproconazole-induced effects on CAR-dependent gene expression, liver weight, and hepatic lipid accumulation were more prominent in wild-type mice, where significant genotype differences were observed at the high-dose level. Moreover, high-dose cyproconazole-treated mice from the wild-type group responded with a marked increase in hepatocellular proliferation, while hCAR/hPXR mice did not. In conclusion, our data demonstrate that cyproconazole and PB induce CAR/PXR downstream effects in hepatocytes in vivo via both, the murine and human receptors. At high doses of cyproconazole, however, the responses were clearly more pronounced in wild-type mice, indicating increased sensitivity of rodents to CAR agonist-induced effects in hepatocytes.

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References

  1. Armeanu-Ebinger S, Wenz J, Seitz G et al (2012) Characterisation of the cell line HC-AFW1 derived from a pediatric hepatocellular carcinoma. PLoS One 7(5):e38223

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. Baan R, Grosse Y, Straif K et al (2009) A review of human carcinogens–Part F: chemical agents and related occupations. Lancet Oncol 10(12):1143–1144

    Article  PubMed  Google Scholar 

  3. Bock KW, Kohle C (2005) Ah receptor- and TCDD-mediated liver tumor promotion: clonal selection and expansion of cells evading growth arrest and apoptosis. Biochem Pharmacol 69(10):1403–1408

    CAS  Article  PubMed  Google Scholar 

  4. Braeuning A (2014) Liver cell proliferation and tumor promotion by phenobarbital: relevance for humans? Arch Toxicol 88(10):1771–1772

    CAS  Article  PubMed  Google Scholar 

  5. Braeuning A, Schwarz M (2016) Is the question of phenobarbital as potential liver cancer risk factor for humans really resolved? Arch Toxicol 90(6):1525–1526

    CAS  Article  PubMed  Google Scholar 

  6. Braeuning A, Kohle C, Buchmann A, Schwarz M (2011) Coordinate regulation of cytochrome P450 1a1 expression in mouse liver by the aryl hydrocarbon receptor and the beta-catenin pathway. Toxicol Sci 122(1):16–25

    CAS  Article  PubMed  Google Scholar 

  7. Braeuning A, Gavrilov A, Brown S, Wolf CR, Henderson CJ, Schwarz M (2014) Phenobarbital-mediated tumor promotion in transgenic mice with humanized CAR and PXR. Toxicol Sci 140(2):259–270

    CAS  Article  PubMed  Google Scholar 

  8. Braeuning A, Thomas M, Hofmann U et al (2015a) Comparative analysis and functional characterization of HC-AFW1 hepatocarcinoma cells: cytochrome P450 expression and induction by nuclear receptor agonists. Drug Metab Dispos 43(11):1781–1787

  9. Braeuning A, Henderson CJ, Wolf CR, Schwarz M (2015b) Model systems for understanding mechanisms of nongenotoxic carcinogenesis: response. Toxicol Sci 147(2):299–300

  10. Currie RA, Peffer RC, Goetz AK, Omiecinski CJ, Goodman JI (2014) Phenobarbital and propiconazole toxicogenomic profiles in mice show major similarities consistent with the key role that constitutive androstane receptor (CAR) activation plays in their mode of action. Toxicology 321:80–88

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. EFSA (2010) Conclusion on the peer review of the pesticide risk assessment of the active substance cyproconazole. EFSA J 8(11):1897

    Article  Google Scholar 

  12. Elcombe CR, Peffer RC, Wolf DC et al (2014) 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 44(1):64–82

    CAS  Article  PubMed  Google Scholar 

  13. Foster JR, Lund G, Sapelnikova S, Tyrrell DL, Kneteman NM (2014) Chimeric rodents with humanized liver: bridging the preclinical/clinical trial gap in ADME/toxicity studies. Xenobiotica 44(2):109–122

    CAS  Article  PubMed  Google Scholar 

  14. Goettel M, Melching-Kollmuss S, Honarvar N, Marxfeld H, Elcombe CR, Fegert I (2015) Mouse liver tumors induced by prochloraz have a CAR-like mode of action and are not relevant to humans. Toxicol Suppl Toxicol Sci 144(1):351

    Google Scholar 

  15. Goetz AK, Dix DJ (2009) Toxicogenomic effects common to triazole antifungals and conserved between rats and humans. Toxicol Appl Pharmacol 238(1):80–89

    CAS  Article  PubMed  Google Scholar 

  16. Halwachs S, Wassermann L, Lindner S, Zizzadoro C, Honscha W (2013) Fungicide prochloraz and environmental pollutant dioxin induce the ABCG2 transporter in bovine mammary epithelial cells by the arylhydrocarbon receptor signaling pathway. Toxicol Sci 131(2):491–501

    CAS  Article  PubMed  Google Scholar 

  17. 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

    CAS  Article  PubMed  Google Scholar 

  18. Holsapple MP, Pitot HC, Cohen SM et al (2006) Mode of action in relevance of rodent liver tumors to human cancer risk. Toxicol Sci 89(1):51–56

    CAS  Article  PubMed  Google Scholar 

  19. LeBaron MJ, Geter DR, Rasoulpour RJ et al (2013) An integrated approach for prospectively investigating a mode-of-action for rodent liver effects. Toxicol Appl Pharmacol 270:164–173

    CAS  Article  PubMed  Google Scholar 

  20. Luisier R, Lempiainen H, Scherbichler N et al (2014) Phenobarbital induces cell cycle transcriptional responses in mouse liver humanized for constitutive androstane and pregnane x receptors. Toxicol Sci 139(2):501–511

    CAS  Article  PubMed  Google Scholar 

  21. Maglich JM, Parks DJ, Moore LB et al (2003) Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes. J Biol Chem 278(19):17277–17283

    CAS  Article  PubMed  Google Scholar 

  22. Mutoh S, Sobhany M, Moore R, et al. (2013) Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling. Sci Signal 6(274):ra31

    Article  PubMed  Google Scholar 

  23. Peffer RC, Moggs JG, Pastoor T et al (2007) Mouse liver effects of cyproconazole, a triazole fungicide: role of the constitutive androstane receptor. Toxicol Sci 99(1):315–325

    CAS  Article  PubMed  Google Scholar 

  24. Petzuch B, Groll N, Schwarz M, Braeuning A (2015) Application of HC-AFW1 hepatocarcinoma cells for mechanistic studies: regulation of cytochrome P450 2B6 expression by dimethyl sulfoxide and early growth response 1. Drug Metab Dispos 43(11):1727–1733

    CAS  Article  PubMed  Google Scholar 

  25. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. Poland A, Mak I, Glover E (1981) Species differences in responsiveness to 1,4-bis[2-(3,5-dichloropyridyloxy)]-benzene, a potent phenobarbital-like inducer of microsomal monooxygenase activity. Mol Pharmacol 20(2):442–450

    CAS  PubMed  Google Scholar 

  27. Rieke S, Koehn S, Hirsch-Ernst K, Pfeil R, Kneuer C, Marx-Stoelting P (2014) Combination effects of (tri)azole fungicides on hormone production and xenobiotic metabolism in a human placental cell line. Int J Environ Res Public Health 11(9):9660–9679

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. Ross J, Plummer SM, Rode A et al (2010) Human constitutive androstane receptor (CAR) and pregnane X receptor (PXR) support the hypertrophic but not the hyperplastic response to the murine nongenotoxic hepatocarcinogens phenobarbital and chlordane in vivo. Toxicol Sci 116(2):452–466

    CAS  Article  PubMed  Google Scholar 

  29. Scheer N, Ross J, Rode A et al (2008) A novel panel of mouse models to evaluate the role of human pregnane X receptor and constitutive androstane receptor in drug response. J Clin Invest 118(9):3228–3239

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Scheer N, Ross J, Kapelyukh Y, Rode A, Wolf CR (2010) In vivo responses of the human and murine pregnane X receptor to dexamethasone in mice. Drug Metab Dispos 38(7):1046–1053. doi:10.1124/dmd.109.031872

    CAS  Article  PubMed  Google Scholar 

  31. 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

    Article  PubMed  Google Scholar 

  32. Schulthess P, Loffler A, Vetter S et al (2015) Signal integration by the CYP1A1 promoter-a quantitative study. Nucl Acids Res 43(11):5318–5330

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Weiss F, Schnabel A, Planatscher H, et al. (2015) Indirect protein quantification of drug-transforming enzymes using peptide group-specific immunoaffinity enrichment and mass spectrometry. Sci Rep 5:8759

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. Whysner J, Ross PM, Williams GM (1996) Phenobarbital mechanistic data and risk assessment: enzyme induction, enhanced cell proliferation, and tumor promotion. Pharmacol Ther 71(1–2):153–191

    CAS  Article  PubMed  Google Scholar 

  35. Yamada T, Okuda Y, Kushida M et al (2014) Human hepatocytes support the hypertrophic but not the hyperplastic response to the murine nongenotoxic hepatocarcinogen sodium phenobarbital in an in vivo study using a chimeric mouse with humanized liver. Toxicol Sci 142(1):137–157

    CAS  Article  PubMed  Google Scholar 

  36. Zukunft J, Lang T, Richter T et al (2005) A natural CYP2B6 TATA box polymorphism (-82T–> C) leading to enhanced transcription and relocation of the transcriptional start site. Mol Pharmacol 67(5):1772–1782

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

The authors greatly acknowledge expert technical assistance by Johanna Mahr, Silvia Vetter, Elke Zabinsky and Barbara Freytag. We also thank Dr. U. Zanger (Stuttgart, Germany) for providing the hCYP2B6 reporter plasmid, Dr. S. Armeanu-Ebinger, Dr. J. Fuchs and Dr. S. Warmann (Tuebingen, Germany) for providing access to HC-AFW1 cells, and Dr. C.R. Wolf (Dundee, UK) for the gift of CYP antisera. This study was supported by the German Federal Institute for Risk Assessment (Grant SFP1322-499).

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Correspondence to Albert Braeuning.

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Marx-Stoelting, P., Ganzenberg, K., Knebel, C. et al. Hepatotoxic effects of cyproconazole and prochloraz in wild-type and hCAR/hPXR mice. Arch Toxicol 91, 2895–2907 (2017). https://doi.org/10.1007/s00204-016-1925-2

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Keywords

  • Liver
  • Constitutive androstane receptor
  • Aryl hydrocarbon receptor
  • Phenobarbital
  • Cytochrome P450
  • Tumor promotion