Amiodarone-mediated thyroid hormone disruption induces mitochondrial apoptosis and G1 arrest during chick embryogenesis.

Thyroid hormones (THs) coordinate proliferation, apoptosis, and tissue remodeling during vertebrate development, yet how TH signaling constrains embryonic cell death in avian embryos remains unclear. Here, we used amiodarone, a pharmacological disruptor of TH signaling with reported effects on TH receptors and deiodinase-mediated hormone metabolism, to perturb TH signaling at the onset of incubation in chick embryos and integrate systems-level and functional readouts. In silico docking predicted favorable binding of amiodarone to all three deiodinase isoforms (DIO1-3), while whole-embryo assays showed a progressive reduction in total embryonic deiodinase capacity from day 2 to day 4. Treated embryos developed lateral plate mesoderm-associated craniofacial, limb, and ventral body-wall defects and exhibited widespread apoptosis, as evidenced by Nile blue sulfate staining, DNA laddering, TUNEL, Annexin V/propidium iodide flow cytometry, and cleaved CASPASE-3 immunolocalization. qRT-PCR and immunoblotting demonstrated downregulation of BCL2 and PCNA with upregulation of BAX, P53, BAD, caspases, and cleaved CASPASE-3, indicating activation of mitochondrial apoptotic signaling accompanied by increased P53 expression. High-resolution proteomics further supported induction of apoptotic and mitochondrial stress-associated pathways alongside suppression of vesicle trafficking and mitochondrial translation proteins. Cell-cycle profiling showed accumulation of cells in sub-G0/G1 and G0/G1 phases, indicating G1 checkpoint arrest and reduced proliferative capacity. Promoter analysis predicted putative thyroid hormone response elements in selected apoptotic regulators, suggesting potential TH-responsive regulatory sites requiring functional validation. Together, these data support an important role for early TH signaling in maintaining the proliferation-apoptosis balance during chick embryonic morphogenesis.