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