title: Embryotoxic and pharmacologic potency ranking of six azoles in the rat whole embryo culture by morphological and transcriptomic analysis.
abstract: Differential gene expression analysis in the rat whole embryo culture (WEC) assay provides mechanistic insight into the embryotoxicity of test compounds. In our study, we hypothesized that comparative analysis of the transcriptomes of rat embryos exposed to six azoles (flusilazole, triadimefon, ketoconazole, miconazole, difenoconazole and prothioconazole) could lead to a better mechanism-based understanding of their embryotoxicity and pharmacological action. For evaluating embryotoxicity, we applied the total morphological scoring system (TMS) in embryos exposed for 48h. The compounds tested showed embryotoxicity in a dose-response fashion. Functional analysis of differential gene expression after 4h exposure at the ID10(effective dose for 10% decreased TMS), revealed the sterol biosynthesis pathway and embryonic development genes, dominated by genes in the retinoic acid (RA) pathway, albeit in a differential way. Flusilazole, ketoconazole and triadimefon were the most potent compounds affecting the RA pathway, while in terms of regulation of sterol function, difenoconazole and ketoconazole showed the most pronounced effects. Dose-dependent analysis of the effects of flusilazole revealed that the RA pathway related genes were already differentially expressed at low dose levels while the sterol pathway showed strong regulation at higher embryotoxic doses, suggesting that this pathway is less predictive for the observed embryotoxicity. A similar analysis at the 24-hour time point indicated an additional time-dependent difference in the aforementioned pathways regulated by flusilazole. In summary, the rat WEC assay in combination with transcriptomics could add a mechanistic insight into the embryotoxic potency ranking and pharmacological mode of action of the tested compounds.
authors.affiliation: Division of Toxicology, Wageningen University, The Netherlands; National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands. Electronic address: myrto.dimopoulou@wur.nl.
authors.forename: Myrto
authors.initials: M
authors.lastname: Dimopoulou
authors.affiliation: National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
authors.forename: Aart
authors.initials: A
authors.lastname: Verhoef
authors.affiliation: National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
authors.forename: Jeroen L A
authors.initials: JLA
authors.lastname: Pennings
authors.affiliation: Division of Toxicology, Wageningen University, The Netherlands; BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany.
authors.forename: Bennard
authors.initials: B
authors.lastname: van Ravenzwaay
authors.affiliation: Division of Toxicology, Wageningen University, The Netherlands.
authors.forename: Ivonne M C M
authors.initials: IMCM
authors.lastname: Rietjens
authors.affiliation: National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands; Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands.
authors.forename: Aldert H
authors.initials: AH
authors.lastname: Piersma
mesh_terms: D000818:Animals; D001393:Azoles / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D046149:Embryo Culture Techniques / Q000379:methods*; D004622:Embryo, Mammalian / Q000187:drug effects* / Q000502:physiology*; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D053263:Gene Regulatory Networks / Q000187:drug effects / Q000235:genetics; D011247:Pregnancy; D051381:Rats; D017208:Rats, Wistar
chemical_list: D001393:Azoles
keywords: Azoles; Embryonic development; Retinoic acid; Sterol biosynthesis; Toxicogenomics; Whole embryo culture