115 | Thyroid-active endocrine disruptors shape distinct neurobehavioral signatures in zebrafish larvae

Tommaso Sivestrini^1|2, Annamaria Iannetta¹, Ludovica Sulcanese¹, Giovanni Angelozzi¹, Vanessa D’Altilia¹, Monica Ceci¹, Monia Perugini¹ | ¹Advanced Study “Theoretical and Applied Neuroscience”, Preclinical clinical and Translational Neuroscience, University of Camerino, Italy; ²Department of Bioscience and Agro-Food and Environmental Technology, University of Teramo, Italy.

Thyroid hormones are essential regulators of vertebrate neurodevelopment, and the hypothalamic–pituitary–thyroid (HPT) axis disruption is one of the a plausible mechanism for early-life related to the production of persistent functional alterations. Here, we used zebrafish (Danio rerio) larvae as an in vivo model to define neurobehavioral fingerprints of thyroid-active endocrine-disrupting chemicals (EDCs), along with anatomical and molecular readouts, with the goal of applying this framework to environmental chemicals. First, we outlined phenotype of larvae exposed to two reference thyroid disruptors, methimazole (MMI) and phenylthiourea (PTU), using an automated VAMR (Visual/Acoustic Motor Response) battery. Behavioral endpoints include baseline locomotion and stimulus-evoked reactivity to standardized light and acoustic stimuli, together with habituation dynamics across repeated stimulus blocks. To test mechanistic specificity, we performed a T3 rescue by co-exposing triiodothyronine and reference chemical to check reversal or attenuation of the behavioral phenotype and complemented by quantification of thyroid hormones (T3 and T4) to directly confirm thyroid perturbation under the exposure conditions. Second, we extend the same behavioral pipeline to a set of environmental chemicals prioritized through US EPA CompTox/ToxCast screening for thyroid-relevant targets and filtered for sublethal exposure concentration using Fish Embryo Acute Toxicity (FET) Test. Behavioral signatures across concentrations are then integrated with neuroanatomical measurements (e.g., head/eye morphometrics) and transcriptional markers relevant to thyroid signaling and neurodevelopment to compare each chemical’s profile to the reference signature and then cluster chemicals into similar groups. Overall, this work aims to deliver a reproducible target-to-behavior framework for thyroid-active EDCs, enabling prioritization of chemicals and strengthening evidence for risk assessment strategies focused on vulnerable windows of brain development.