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Difenoconazole was believed to induce a large suite of symptoms during zebrafish development, but little is known about the negative invisible effect known as endocrine disruption. In this study, zebrafish ( Danio rerio ) embryos were exposed to various concentrations of difenoconazole from fertilization to 120 h post-fertilization (hpf), and the whole body content of thyroid hormone and gene transcription in the hypothalamic–pituitary–thyroid (HPT) axis were investigated. Results showed thyroxine (T4) levels were significantly decreased, while triiodothyronine (T3) concentrations were not changed. Moreover, the mRNA transcription of corticotrophin-releasing hormone ( crh ), thyroid-stimulating hormone ( tshβ ), transthyretin ( ttr ), thyronine deiodinase ( dio1 and dio2 ), uridine diphosphate glucuronosyltransferase ( ugt1ab ) in the HPT axis were significantly up-regulated, but the transcriptions of thyroglobulin ( tg ), sodium/iodide symporter ( nis ) and thyroid hormone receptors trβ were not changed. The overall results showed that exposure to difenoconazole could alter thyroid hormone levels and gene transcription in zebrafish larvae, indicating thyroid endocrine disruption. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA) can regulate the expression of its target gene by mediating mRNA cleavage or by translational repression at a post-transcriptional level. Usually, one miRNA may regulate many genes as its targets, while one gene may also be targeted by many miRNAs. We previously demonstrated that cyb561d2 , whose protein product is involved in cell defense, and chemical stress, is targeted by miR-155 in adult zebrafish ( Danio rerio ) when exposed to fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl) phenyl]-4-[(trifluoromethyl) sulphinyl]-1H-pyrazole-3-carbonitrile). Microcosm Targets prediction showed that the cyb561d2 gene is also highly possibly targeted by miR-194a , miR-216b , miR-429 , and miR-499 . These interactions need to be further validated experimentally. In this study, we evaluated the effects of fipronil on miR-194a , miR-216b , miR-429 , miR-499 and cyb561d2 in zebrafish and investigated whether these four miRNAs c ould regulate the expression of cyb561d2 in both mRNA and protein levels. The expression of cyb561d2 was upregulated in both mRNA and protein level in a dose-dependent manner upon stimulation of fipronil, and miR-216b and miR-499 were downregulated concurrently, whereas there was no significant changes were observed in the expression level of miR-194a and miR-429 . The dual luciferase report assay demonstrated that miR-216 b and miR-499 interacted with cyb561d2 3′-untranslated regions (3′-UTR), miR-194a and miR-429 did not stimulate degradation of cyb561d2 mRNA. The expression of cyb561d2 was reduced in both mRNA and protein level when ZF4 cells were transfected with miR-499 mimic, whereas expression level of both mRNA and protein was increased when endogenous miR-499 was inhibited by transfection with miR-499 inhibitor. Likewise, the mRNA and protein level of cyb561d2 was affected by treatment with the mimics and the inhibitor of miR-216b . In contrast, when ZF4 cells were transfected with a mimic of miR-194a or miR-429 , the expression of cyb561d2 mRNA was not significantly changed. As a result, cyb561d2 is targeted by miR-155 , miR-216b and miR-499 upon fipronil exposure, and miR-194a and miR-429 can not target cyb561d2 . The expression pattern of these 3 miRNAs presents novel fipronil responses that could be used as a toxicological biomarker. [ABSTRACT FROM AUTHOR]