3'DGE-seq analysis of fetal rat testis reveals limited transcriptional changes after exposure to azole fungicides triticonazole and flusilazole despite adverse in vivo effects
Purpose: Azoles are widely used antifungal agents. However, many azoles have been shown to have endocrine disrupting properties including triticonazole and flusilazole that we previously showed have adverse effects in vivo. The purpose of this study was to elaborate on how flusilazole and triticonazole can disrupt male reproductive development by different mechanisms, and to investigate whether feminization effects such as short AGD in males can also be detected at the transcript level in fetal testes. Methods: Testicular mRNA profiles of fetal rats at gestational day 17 or 21 exposed to vehicle, flusilazole, and triticonazole during gestation (from day 7-21) were generated by 3'digital gene expression sequencing, qRT–PCR validation was performed using TaqMan assays and testis histology was assesed by H&E staining and bright field microscopy, protein expression was assesed by immunofluorescence staining and fluorescence microcopy. Results: We identified 17,350 transcripts in the fetal rat testes. 9 transcripts showed differential expression between control and flusilazole or triticonazole exposed rats, with a fold change =1.5 and p value <0.05. Altered expression of 2 genes was confirmed with qRT–PCR. Testis histology was normal. Conclusions: Sequencing revealed few transcriptional changes in the fetal rat testes exposed to flusilazole or triticonazole. This suggests that flusilazole may be directly targeting steroidogenic enzyme activity in the testis at the protein level, whereas triticonazole may primarily disturbe androgen signaling in androgen-sensitive tissues. Expression of Calb2 and Gsta2 was altered by flusilazole but not triticonazole and may pinpoint novel pathways of disrupted testicular steroid synthesis. Our findings have wider implication for how we integrate omics data in chemical testing frameworks, including selection of non-animal test methods and building of Adverse Outcome Pathways for regulatory purposes. Overall design: Testicular mRNA profiles of fetal rats at gestational day 17 or 21 exposed to vehicle, flusilazole, and triticonazole during gestation (from day 7-21) were generated by 3'digital gene expression sequencing.