Unveiling the role of Hedgehog signaling pathway in mitigating methylisothiazolinone-induced anxiety in zebrafish

Methylisothiazolinone (MIT), a prevalent antimicrobial agent utilized in pharmaceuticals, cosmetics, and industrial contexts, presents potential toxicity concerns. Recent investigations have corroborated its neurotoxic effects in zebrafish and suggested involvement of the Hedgehog signaling pathway, although the specific regulatory mechanisms remain to be elucidated. In this study, transcriptome-based gene-pathway network analysis identified hhip within the Hedgehog pathway. Expression profiling and in situ hybridization revealed it is suppressed by MIT exposure and displays high expression in the zebrafish midbrain and hypothalamus. Dual-luciferase reporter assays in the zebrafish Pac2 cell line demonstrated that hhip suppresses transcription of the Hedgehog target gene gli1 and modulates reactive oxygen species (ROS) levels and oxidative stress-related gene expression. Moreover, pharmacological inhibition of the Hedgehog pathway with Ciliobrevin A (CA) during MIT exposure ameliorated anxiety-like behaviors in zebrafish, such as aberrant edge preference (thigmotaxis). This intervention downregulated Hedgehog pathway activity, elevated expression of oxidative stress genes (e.g., gpx4), reduced expression of the ferroptosis marker gene tf, attenuated the loss of Nissl bodies in brain tissue, and decreased brain iron content. These findings suggested that MIT exposure is associated with molecular signatures indicative of Hedgehog pathway activation. This cascade of molecular events is associated with subsequent anxiety-like behaviors, implying its potential contribution to the behavioral phenotype. In vitro studies using Pac2 cells further corroborated that inhibition of the Hedgehog pathway alleviates the increase in lipid peroxidation induced by the ferroptosis inducer Erastin. Collectively, these results elucidated the molecular regarding MIT-induced neurotoxicity and offer potential preventive and interventional strategies for addressing toxicity associated with this class of compounds.