Transcriptome analysis of fetal rat testis following intrauterine exposure to the azole fungicides triticonazole and flusilazole reveals subtle changes despite adverse endocrine effects

Azoles are used in agriculture and medicine to combat fungal infections. We have previously examined the endocrine disrupting properties of the agricultural azole fungicides triticonazole and flusilazole. Triticonazole displayed strong androgen receptor (AR) antagonism in vitro, whereas in utero exposure resulted in anti-androgenic effects in vivo evidenced by shorter anogenital distance (AGD) in fetal male rats. Flusilazole displayed strong AR antagonism, but less potent than triticonazole, and disrupted steroidogenesis in vitro, whereas in utero exposure disrupted fetal male plasma hormone levels. To elaborate on how these azole fungicides 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, we profiled fetal testis transcriptomes after in utero exposure to triticonazole and flusilazole by 3′Digital Gene Expression (3′DGE). The analysis revealed few transcriptional changes after exposure to either compound at gestation day 17 and 21. This suggests that the observed influence of flusilazole on hormone production may be by directly targeting steroidogenic enzyme activity in the testis at the protein level, whereas observations of shorter AGD by triticonazole may primarily be due to disturbed 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.