Skip to main content

Hepatotoxic effects of (tri)azole fungicides in a broad dose range

Abstract

The toxicological relevance of effects observed at molecular stage, which occur at dose levels well below classical no-observed adverse effect levels is currently subject to controversial scientific debate. While the importance of molecular effects for the identification of a mode of action or an adverse outcome pathway is undisputed, their impact for other regulatory purposes remains uncertain. Here, we report the results of a 28-day rat-feeding study including three widely used hepatotoxic (tri)azole fungicides (cyproconazole, epoxiconazole and prochloraz) administered individually at five dose levels, ranging from slightly above the reference values to a clear toxic effect dose. Parameters analysed included pathology, histopathology, clinical chemistry and particularly effects on the molecular level. Since azole fungicides are considered to cause liver toxicity by a mechanism involving the constitutive androstane receptor (CAR), a known CAR activator (phenobarbital, PB) was administered to investigate potential similarities between triazoles and PB-mediated liver toxicity by pathway-focused gene expression analysis. Our results show an increase in liver weights and additionally histopathological changes (hepatocellular hypertrophy) for all substances at the top dose levels. The effects on liver weight were most pronounced for cyproconazole by which also the animals receiving the next lower dose were affected. In addition, vacuolisation of hepatocytes was observed at the top dose level. No such findings were obtained with any substance at lower doses to which consumers and operators might be exposed to. In contrast, the expression of sensitive marker genes (like some cytochrome-P-450 isoforms) was significantly affected also at the lower dose levels. While some of these changes, like the induction of genes related to fatty acid and phospholipid metabolism (e.g. Fasn, Fat/Cd36, Ppargc1a) or xenobiotic metabolism (Cyp1a1, Cyp2b1, Cyp3a2), could be associated with high dose effects like hepatocellular vacuolisation or hypertrophy, a histopathological correlate was lacking for others.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  • Amaral JD, Xavier JM, Steer CJ, Rodrigues CM (2010) The role of p53 in apoptosis. Discov Med 9:145–152

    PubMed  Google Scholar 

  • Beausoleil C, Ormsby JN, Gies A, Hass U, Heindel JJ, Holmer ML, Nielsen PJ, Munn S, Schoenfelder G (2013) Low dose effects and non-monotonic dose responses for endocrine active chemicals: science to practice workshop: workshop summary. Chemosphere 93:847–856

    CAS  Article  PubMed  Google Scholar 

  • Boobis AR, Doe JE, Heinrich-Hirsch B, Meek ME, Munn S, Ruchirawat M, Schlatter J, Seed J, Vickers C (2008) IPCS framework for analyzing the relevance of a noncancer mode of action for humans. Crit Rev Toxicol 38:87–96

    CAS  Article  PubMed  Google Scholar 

  • OECD (2008) Repeated dose 28-day oral toxicity study in rodents. OECD, OECD Guideline For The Testing Of Chemicals, Paris

    Book  Google Scholar 

  • 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

  • Dewhurst I, Dellarco V (2004) Propiconazole. Joint WHO/FAO meeting on pesticide residues. Pesticide Residues in Food Evaluations Part II Toxicological Evaluation

  • Edwards SG, Godley NP (2010) Reduction of Fusarium head blight and deoxynivalenol in wheat with early fungicide applications of prothioconazole. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 27:629–635

    CAS  Article  PubMed  Google Scholar 

  • EFSA (2008a) Conclusion on the peer review of epoxiconazole. EFSA Scientific Report 138:1–80

    Google Scholar 

  • EFSA (2008b) Conclusion on the peer review of the pesticide risk assessment of the active substance tebuconazole. EFSA Scientific Report 176:1–109

    Google Scholar 

  • Goetz AK, Dix DJ (2009a) Mode of action for reproductive and hepatic toxicity inferred from a genomic study of triazole antifungals. Toxicol Sci 110:449–462

    CAS  Article  PubMed  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  • Goetz AK, Bao W, Ren H, Schmid JE, Tully DB, Wood C, Rockett JC, Narotsky MG, Sun G, Lambert GR, Thai SF, Wolf DC, Nesnow S, Dix DJ (2006) Gene expression profiling in the liver of CD-1 mice to characterize the hepatotoxicity of triazole fungicides. Toxicol Appl Pharmacol 215:274–284. doi:10.1016/j.taap.2006.02.016

    CAS  Article  PubMed  Google Scholar 

  • Goetz AK, Rockett JC, Ren H, Thillainadarajah I, Dix DJ (2009) Inhibition of rat and human steroidogenesis by triazole antifungals. Syst Biol Reprod Med 55:214–226

    CAS  Article  PubMed  Google Scholar 

  • Handschin C (2009) The biology of PGC-1alpha and its therapeutic potential. Trends Pharmacol Sci 30:322–329

    CAS  Article  PubMed  Google Scholar 

  • Hester SD, Wolf DC, Nesnow S, Thai SF (2006) Transcriptional profiles in liver from rats treated with tumorigenic and non-tumorigenic triazole conazole fungicides: propiconazole, triadimefon, and myclobutanil. Toxicol Pathol 34:879–894

    CAS  Article  PubMed  Google Scholar 

  • Hester S, Moore T, Padgett WT, Murphy L, Wood CE, Nesnow S (2012) The hepatocarcinogenic conazoles: cyproconazole, epoxiconazole, and propiconazole induce a common set of toxicological and transcriptional responses. Toxicol Sci 127:54–65

    CAS  Article  PubMed  Google Scholar 

  • IARC (2001) Phenobarbital and its sodium salt, vol 79. IARC Press. IARC Monographs, Lyon, pp 161–288

    Google Scholar 

  • Jacobsen PR, Axelstad M, Boberg J, Isling LK, Christiansen S, Mandrup KR, Berthelsen LO, Vinggaard AM, Hass U (2012) Persistent developmental toxicity in rat offspring after low dose exposure to a mixture of endocrine disrupting pesticides. Reprod Toxicol 34:237–250

    CAS  Article  PubMed  Google Scholar 

  • Leone TC, Lehman JJ, Finck BN, Schaeffer PJ, Wende AR, Boudina S, Courtois M, Wozniak DF, Sambandam N, Bernal-Mizrachi C, Chen Z, Holloszy JO, Medeiros DM, Schmidt RE, Saffitz JE, Abel ED, Semenkovich CF, Kelly DP (2005) PGC-1alpha deficiency causes multi-system energy metabolic derangements: muscle dysfunction, abnormal weight control and hepatic steatosis. PLoS Biol 3:e101. doi:10.1371/journal.pbio.0030101

    PubMed Central  Article  PubMed  Google Scholar 

  • Long M, Laier P, Vinggaard AM, Andersen HR, Lynggaard J, Bonefeld-Jorgensen EC (2003) Effects of currently used pesticides in the AhR-CALUX assay: comparison between the human TV101L and the rat H4IIE cell line. Toxicology 194:77–93

    CAS  Article  PubMed  Google Scholar 

  • Menegola E, Broccia ML, Di RF, Giavini E (2006) Postulated pathogenic pathway in triazole fungicide induced dysmorphogenic effects. Reprod Toxicol 22:186–195

    CAS  Article  PubMed  Google Scholar 

  • Myers JP, vom Saal FS, Akingbemi BT, Arizono K, Belcher S, Colborn T, Chahoud I, Crain DA, Farabollini F, Guillette LJ Jr, Hassold T, Ho SM, Hunt PA, Iguchi T, Jobling S, Kanno J, Laufer H, Marcus M, McLachlan JA, Nadal A, Oehlmann J, Olea N, Palanza P, Parmigiani S, Rubin BS, Schoenfelder G, Sonnenschein C, Soto AM, Talsness CE, Taylor JA, Vandenberg LN, Vandenbergh JG, Vogel S, Watson CS, Welshons WV, Zoeller RT (2009) Why public health agencies cannot depend on good laboratory practices as a criterion for selecting data: the case of bisphenol A. Environ Health Perspect 117:309–315

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  • Nesnow S, Ward W, Moore T, Ren H, Hester SD (2009) Discrimination of tumorigenic triazole conazoles from phenobarbital by transcriptional analyses of mouse liver gene expression. Toxicol Sci 110:68–83

    CAS  Article  PubMed  Google Scholar 

  • OECD (2013) Guidance document on developing and assessing adverse outcome pathways. Series on testing and assessment 184. OECD, OECD Environment, Health and Safety Publications, Paris

    Google Scholar 

  • Peffer RC, Moggs JG, Pastoor T, Currie RA, Wright J, Milburn G, Waechter F, Rusyn I (2007) Mouse liver effects of cyproconazole, a triazole fungicide: role of the constitutive androstane receptor. Toxicol Sci 99:315–325

    CAS  Article  PubMed  Google Scholar 

  • Rhomberg LR, Goodman JE (2012) Low-dose effects and nonmonotonic dose-responses of endocrine disrupting chemicals: has the case been made? Regul Toxicol Pharmacol 64:130–133

    CAS  Article  PubMed  Google Scholar 

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

    Google Scholar 

  • SA EF (2011) Conclusion on the peer review of the risk assessment of the active substance prochloraz. EFSA J 9(7):2323

    Google Scholar 

  • Sun G, Thai SF, Tully DB, Lambert GR, Goetz AK, Wolf DC, Dix DJ, Nesnow S (2005) Propiconazole-induced cytochrome P450 gene expression and enzymatic activities in rat and mouse liver. Toxicol Lett 155:277–287

    CAS  Article  PubMed  Google Scholar 

  • Sun G, Grindstaff RD, Thai SF, Lambert GR, Tully DB, Dix DJ, Nesnow S (2007) Induction of cytochrome P450 enzymes in rat liver by two conazoles, myclobutanil and triadimefon. Xenobiotica 37:180–193

    CAS  Article  PubMed  Google Scholar 

  • Tamura K, Inoue K, Takahashi M, Matsuo S, Irie K, Kodama Y, Ozawa S, Nishikawa A, Yoshida M (2013) Dose-response involvement of constitutive androstane receptor in mouse liver hypertrophy induced by triazole fungicides. Toxicol Lett 221:47–56

    CAS  Article  PubMed  Google Scholar 

  • Trosken ER, Scholz K, Lutz RW, Volkel W, Zarn JA, Lutz WK (2004) Comparative assessment of the inhibition of recombinant human CYP19 (aromatase) by azoles used in agriculture and as drugs for humans. Endocr Res 30:387–394

    Article  PubMed  Google Scholar 

  • Trosken ER, Adamska M, Arand M, Zarn JA, Patten C, Volkel W, Lutz WK (2006) Comparison of lanosterol-14 alpha-demethylase (CYP51) of human and Candida albicans for inhibition by different antifungal azoles. Toxicology 228:24–32

    Article  PubMed  Google Scholar 

  • Tully DB, Bao W, Goetz AK, Blystone CR, Ren H, Schmid JE, Strader LF, Wood CR, Best DS, Narotsky MG, Wolf DC, Rockett JC, Dix DJ (2006) Gene expression profiling in liver and testis of rats to characterize the toxicity of triazole fungicides. Toxicol Appl Pharmacol 215:260–273

    CAS  Article  PubMed  Google Scholar 

  • Vanden Bossche H, Willemsens G, Janssen PA (1988) Cytochrome-P-450-dependent metabolism of retinoic acid in rat skin microsomes: inhibition by ketoconazole. Skin Pharmacol 1:176–185

    CAS  Article  PubMed  Google Scholar 

  • Vandenberg LN, Colborn T, Hayes TB, Heindel JJ, Jacobs DR Jr, Lee DH, Shioda T, Soto AM, vom Saal FS, Welshons WV, Zoeller RT, Myers JP (2012) Hormones and endocrine-disrupting chemicals: low-dose effects and nonmonotonic dose responses. Endocr Rev 33:378–455

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  • Vinken M (2013) The adverse outcome pathway concept: a pragmatic tool in toxicology. Toxicology 312:158–165

    CAS  Article  PubMed  Google Scholar 

  • Ward WO, Delker DA, Hester SD, Thai SF, Wolf DC, Allen JW, Nesnow S (2006) Transcriptional profiles in liver from mice treated with hepatotumorigenic and nonhepatotumorigenic triazole conazole fungicides: propiconazole, triadimefon, and myclobutanil. Toxicol Pathol 34:863–878

    CAS  Article  PubMed  Google Scholar 

  • Wolf DC, Allen JW, George MH, Hester SD, Sun G, Moore T, Thai SF, Delker D, Winkfield E, Leavitt S, Nelson G, Roop BC, Jones C, Thibodeaux J, Nesnow S (2006) Toxicity profiles in rats treated with tumorigenic and nontumorigenic triazole conazole fungicides: propiconazole, triadimefon, and myclobutanil. Toxicol Pathol 34:895–902

    CAS  Article  PubMed  Google Scholar 

  • Zarn JA, Bruschweiler BJ, Schlatter JR (2003) Azole fungicides affect mammalian steroidogenesis by inhibiting sterol 14 alpha-demethylase and aromatase. Environ Health Perspect 111:255–261

    PubMed Central  CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Daniela Neubert, Sabine Krönke, Barbara Freytag and Brigitte Ladwig for excellent technical assistance and Matthias Peiser for the critical revision of the manuscript.

Conflict of interest

The authors do not have any conflict of interest.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to P. Marx-Stoelting.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 229 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Heise, T., Schmidt, F., Knebel, C. et al. Hepatotoxic effects of (tri)azole fungicides in a broad dose range. Arch Toxicol 89, 2105–2117 (2015). https://doi.org/10.1007/s00204-014-1336-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00204-014-1336-1

Keywords

  • Liver
  • Low dose
  • Phenobarbital
  • Triazole
  • Adverse outcome pathway