Azole-Induced Color Vision Deficiency Associated with Thyroid Hormone Signaling: An Integrated in Vivo, in Vitro, and in Silico Study

Azoles that are used in pesticides, pharmaceuticals, and personal care products can have toxic effects on fish. However, there is no information regarding azole-induced visual disorder associated with thyroid disruption. We evaluated changes in retinal morphology, optokinetic response, transcript abundance of the genes involved in color perception and hypothalamic-pituitary-thyroid (HPT) axis, and thyroid hormone (TH) levels in zebrafish larvae exposed to common azoles, such as climbazole (CBZ, 0.1 and 10 μg/L) and triadimefon (TDF, 50 and 500 μg/L), at environmentally relevant and predicted worst-case environmental concentrations. Subsequently, the effect of azoles on TH-dependent GH3 cell proliferation and thyroid receptor (TR)-regulated transcriptional activity, as well as the in silico binding affinity between azoles and TR isoforms, was investigated. Azole exposure decreased cell densities of the ganglion cell layer, inner nuclear layer, and photoreceptor layer. Zebrafish larvae exposed to environmentally relevant concentrations of CBZ and TDF showed a decrease in optokinetic response to green-white and red-white stripes but not blue-white stripes, consistent with disturbance in the corresponding opsin gene expression. Azole exposure also reduced triiodothyronine levels and concomitantly increased HPT-related gene expression. Molecular docking analysis combined with in vitro TR-mediated transactivation and dual-luciferase reporter assays demonstrated that CBZ and TDF exhibited TR antagonism. These results are comparable to those obtained from a known TR antagonist, namely, TR antagonist 1, as a positive control. Therefore, damage to specific color perception by azoles appears to result from lowered TH signaling, indicating the potential threat of environmental TH disruptors to the visual function of fish.