Toxicogenomic effects common to triazole antifungals and conserved between rats and humans

https://doi.org/10.1016/j.taap.2009.04.016Get rights and content

Abstract

The triazole antifungals myclobutanil, propiconazole and triadimefon cause varying degrees of hepatic toxicity and disrupt steroid hormone homeostasis in rodent in vivo models. To identify biological pathways consistently modulated across multiple timepoints and various study designs, gene expression profiling was conducted on rat livers from three separate studies with triazole treatment groups ranging from 6 h after a single oral gavage exposure, to prenatal to adult exposures via feed. To explore conservation of responses across species, gene expression from the rat liver studies were compared to in vitro data from rat and human primary hepatocytes exposed to the triazoles. Toxicogenomic data on triazoles from 33 different treatment groups and 135 samples (microarrays) identified thousands of probe sets and dozens of pathways differentially expressed across time, dose, and species — many of these were common to all three triazoles, or conserved between rodents and humans. Common and conserved pathways included androgen and estrogen metabolism, xenobiotic metabolism signaling through CAR and PXR, and CYP mediated metabolism. Differentially expressed genes included the Phase I xenobiotic, fatty acid, sterol and steroid metabolism genes Cyp2b2 and CYP2B6, Cyp3a1 and CYP3A4, and Cyp4a22 and CYP4A11; Phase II conjugation enzyme genes Ugt1a1 and UGT1A1; and Phase III ABC transporter genes Abcb1 and ABCB1. Gene expression changes caused by all three triazoles in liver and hepatocytes were concentrated in biological pathways regulating lipid, sterol and steroid homeostasis, identifying a potential common mode of action conserved between rodents and humans. Modulation of hepatic sterol and steroid metabolism is a plausible mode of action for changes in serum testosterone and adverse reproductive outcomes observed in rat studies, and may be relevant to human risk assessment.

Introduction

Myclobutanil, propiconazole and triadimefon are agrichemical fungicides with a 1,2,4-N-substituted triazole moiety that binds the heme portion of fungal cyp51, inhibiting fungal lanosterol-14α-demethylase activity, blocking ergosterol biosynthesis and thus controlling several species and strains of fungi (Ghannoum and Rice, 1999, Vanden Bossche et al., 1990). All three of the triazoles in this study are used in the control of brown patch, dollar spot, rust, and several other fungal and plant diseases to protect turf, fruit, and vegetable and seed commodities.

Triazole fungicides exhibit a range of toxicological properties in mammalian species. Effects reported from triazole exposures in rodents include male infertility for myclobutanil and triadimefon in rats, liver tumorigenicity for propiconazole and triadimefon in mice, thyroid tumors for triadimefon in rats, and some measure of reproductive and/or hepatic toxicity for all three of these triazoles (Goetz et al., 2007, U.S. Environmental Protection Agency, 1995, U.S. Environmental Protection Agency, 2005a, U.S. Environmental Protection Agency, 2005b, U.S. Environmental Protection Agency, 2006). Studies examining developmental and reproductive effects of myclobutanil, propiconazole, and triadimefon exposures beginning in gestation and continuing to adulthood in rats have demonstrated that all three triazoles caused increased serum testosterone levels (Goetz et al., 2007). Several studies in mice and rats on the hepatic and thyroid toxicity of triazole fungicides have identified modes of action, in some cases conserved across rodent species, and possibly relevant to the assessment of risk to human health (Allen et al., 2006, Wolf et al., 2006).

Genomic data from these and other studies have linked triazole specific toxicological endpoints to demonstrate the ability of high-content biology to delineate potential pathways of toxicity (Goetz et al., 2006, Hester et al., 2006, Hester and Nesnow, 2008, Martin et al., 2007, Tully et al., 2006, Ward et al., 2006). To date, in vivo gene expression profiles have demonstrated that triazoles appear to modulate CAR and PXR, and subsequently perturb hepatic lipid, sterol, and steroid and xenobiotic metabolism pathways. The concordance of these in vivo observations and gene expression findings demonstrated the ability of genomics to identify potential modes of action and toxicity pathways.

In the present study, genomic data from a series of in vivo and in vitro studies on three triazoles (myclobutanil, propiconazole and triadimefon) were analyzed to test the hypothesis that there are conserved hepatic biological pathways active in rat liver and human hepatocytes that are commonly perturbed by this chemical class. While changes in individual genes did vary between the in vivo and in vitro studies, comparison of homologous genes across species and mapping these to biological pathways facilitated meaningful evaluations of functional significance. These differentially expressed genes and affected pathways were then related to the toxicological endpoints from these same in vivo and in vitro studies, creating a framework for understanding the toxicity pathways common to triazoles and conserved in rats and humans.

Section snippets

Prenatal to adult rat study

Details of the animal husbandry and study design for the prenatal to adult rat study have been previously published (Goetz et al., 2007). For this and all other studies; animal care, handling, and treatment at EPA or by its contractors was conducted in American Association for Accreditation of Laboratory Animal Care — International accredited facilities, and all procedures were approved by an Institutional Animal Care and Use Committee. For the prenatal to adult rat study conducted at EPA,

Hepatocyte cytotoxicity

Overall, there was only significant cell lysis for one triazole treatment group, and for the majority of rat and human samples the percent cytotoxicity was 1% or less (Fig. 1). There was an increase in cytotoxicity for one rat sample following 100 μM propiconazole treatment, but not the other two rat samples in that treatment group. An increase in cytotoxicity was observed for the human 100 μM propiconazole treatment group, to approximately a 27% level.

Serum testosterone levels in vivo

In the Gene Logic repeated dose oral

Discussion

The goal of this study was to compare rat in vivo and in vitro models, to human in vitro models and to identify common, consistent, and conserved gene expression changes that better characterized the modes of action for triazole toxicities. The potential of short term in vivo or in vitro assays for predicting longer-term effects was also explored in this comparative toxicogenomic analysis. Measuring changes in gene expression across different exposure periods and doses, triazole chemicals, and

Acknowledgments

The authors thank Drs Wenjun Bao and Russ Wolfinger (SAS Inc., Cary, NC) for expert advice on data analysis; and Drs Hongzu Ren (EPA) and Stephen Ferguson (CellzDirect Inc., Durham, NC) for excellent technical support. We also thank Dr Douglas Wolf (EPA/ORD) for technical review of this manuscript; and Ms. Jennifer Hill for excellent management of the EPA contracts with Expression Analysis Inc. (Durham, NC), and CellzDirect. Microarrays and reagents for a portion of this study were provided by

References (24)

  • HonkakoskiP. et al.

    Review article: regulation of cytochrome P450 (CYP) genes by nuclear receptors

    J. Biochem.

    (2000)
  • JigorelE. et al.

    Functional expression of sinusoidal drug transporters in primary human and rat hepatocytes

    Drug Metab. Dispos.

    (2005)
  • Cited by (60)

    • “Fishcide” effect of the fungicide difenoconazole in freshwater fish (Labeo rohita): A multi-endpoint approach

      2023, Science of the Total Environment
      Citation Excerpt :

      Likewise, 48 h LC50 value of difenoconazole to zebrafish was calculated as 1.41 mg L−1 (Sanches et al., 2017). Generally, triazole fungicides act by influencing lipid biosynthesis and metabolism (Goetz and Dix, 2009; Hermsen et al., 2011). In the present study, the mortality of fish during bioassay treatment may be due to the accumulation of difenoconazole or its metabolites in the vital organs such as the liver and kidney, which may affect the physiology or metabolism of fish.

    • The developmental toxicity and transcriptome analyses of zebrafish (Danio rerio) embryos exposed to carbon nanoparticles

      2022, Ecotoxicology and Environmental Safety
      Citation Excerpt :

      In fish eggs, the endogenous lipid reserves, mainly phospholipids and triglycerols, were in the form of yolk globules (Biology and Fisheries, 1996). Effect on yolk sac might also be due to modifications in lipid synthesis and metabolism (Goetz and Dix, 2009). Any impairment of the yolk sac could block nutrient supply during embryonic development that could be the reason for abnormal development (Kuder and Gundala, 2018).

    • Multi-level analysis of exposure to triazole fungicides through treated seed ingestion in the red-legged partridge

      2020, Environmental Research
      Citation Excerpt :

      These three genes were also overexpressed in fungicide-exposed partridges in the present study. In other experiments, triazole fungicides induced the overexpression of genes that did not respond in our study (e.g. CYP51 in rat embryo cultures, Robinson et al., 2012; FDFT1, IDI1, SQLE, HMGCR3, HMGCS1 and SC5D in rat embryo cultures, Dimopoulou et al., 2017a; HMGCR and SQLE genes in liver and testis of rats exposed from gestation day six to postnatal day 92, Goetz and Dix, 2009). Some of the genes seemed especially sensitive and were altered by several treatments in exposed partridges.

    View all citing articles on Scopus

    Disclaimer: The United States Environmental Protection Agency through its Office of Research and Development funded and managed the research described here. It has been subjected to Agency administrative review and approved for publication.

    View full text