Abstract
Conazole fungicides such as epoxiconazole are mostly used on cereals of crops to inhibit fungal growth through direct inhibition of sterol 14α-demethylase (CYP51A1). However, this enzyme is highly conserved and in humans it is part of the steroid hormone biosynthesis pathway. Endocrine disrupting effects of epoxiconazole have been shown in rodents and have been substantiated by in vitro data, however, the underlying molecular mechanisms are not clear. We took advantage of a human stem cell based in vitro model for developmental toxicity to study the molecular effects of epoxiconazole. This model is based on 3D cultures of embryoid bodies and differentiation into cardiomyocytes, which mimics the early stages of embryonic development. We have previously shown that epoxiconazole impairs differentiation of these embryoid bodies and therefore has the potential to affect human embryonic development. We employed global transcriptome analysis using RNA sequencing and found that the steroid biosynthesis pathway including CYP51A1, the human sterol 14α-demethylase, was highly deregulated by epoxiconazole in our model. We confirmed that most genes of the steroid biosynthesis pathway were upregulated, including CYP51A1, suggesting a compensatory mechanism at the gene expression level. Our data suggest that epoxiconazole acts mainly by decreasing cholesterol biosynthesis in the cells. We conclude that epoxiconazole bears the potential to harm human embryonic development through inhibition of the steroid biosynthesis pathway. As this may be a common feature of compounds that target sterol 14α-demethylase, we add evidence to the assumption that conazole fungicides may be human developmental toxicants.
Highlights
Studying molecular mechanisms underlying the adverse effects of chemicals can be challenging, especially in the case of developmental toxicity
Our results indicate that epoxiconazole disrupts cholesterol synthesis in human cells, which supports the human relevance of the well-known endocrine disrupting effects shown in rodents
We investigated an early and a late time point of cardiomyocyte differentiation, namely day 2 (D2) and day 7 (D7)
Summary
Studying molecular mechanisms underlying the adverse effects of chemicals can be challenging, especially in the case of developmental toxicity. In vitro models are a good option for studying human developmental toxicity and the causative molecular mechanisms (Liu et al, 2017; Zink et al, 2020). Human induced pluripotent stem cells (hiPSC) can be used to model early embryonic development by formation of embryoid bodies (EBs), in which cells can differentiate into early cell types, such as cardiomyocytes (Liu et al, 2017; Takahashi et al, 2007). We have previously shown that hiPSC differentiating into cardiomyocytes using an eight-day protocol can detect develop mental toxicity of thalidomide. We detected the pesticide epoxiconazole as a positive in our assay, suggesting its known adverse effects in rodents might happen in the human (Lauschke et al, 2020)
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