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Amiodarone-mediated thyroid hormone disruption induces mitochondrial apoptosis and G1 arrest during chick
Juhi Vaishnav1, Bhavana Balakrishnan2, Suresh Balakrishnan3
1Dr. Vikram Sarabhai Institute of Cell and Molecular Biology, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, 390002, India.
Abstract:
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.
Insights
Thyroid hormones (THs) are crucial for embryonic development, controlling cell death and proliferation. Disrupting TH signaling in chick embryos with amiodarone led to developmental defects and increased apoptosis, highlighting THs
Area of Science:
- Developmental Biology
- Endocrinology
- Molecular Biology
Background:
- Thyroid hormones (THs) regulate key processes in vertebrate development, including proliferation and apoptosis.
- The precise role of TH signaling in preventing embryonic cell death, particularly in avian embryos, remains largely unknown.
Purpose of the Study:
- To investigate the role of TH signaling in constraining embryonic cell death during chick development.
- To analyze the effects of disrupting TH signaling on embryonic morphogenesis and cellular processes.
Main Methods:
- Utilized amiodarone, a pharmacological disruptor of TH signaling, in chick embryos.
- Integrated in silico docking, whole-embryo assays, Nile blue sulfate staining, DNA laddering, TUNEL, flow cytometry, qRT-PCR, immunoblotting, proteomics, and cell-cycle profiling.
Main Results:
- Amiodarone disrupted TH signaling, reduced deiodinase activity, and induced widespread apoptosis and developmental defects (craniofacial, limb, ventral body wall).
- Molecular analysis revealed altered expression of apoptosis-related genes (e.g., BCL2, BAX, P53, caspases) and cell cycle arrest.
- Proteomics indicated activation of apoptotic and mitochondrial stress pathways and suppression of vesicle trafficking and mitochondrial translation.
Conclusions:
- Early TH signaling is essential for maintaining the balance between proliferation and apoptosis during chick embryonic development.
- Disruption of TH signaling leads to increased cell death and morphological abnormalities, underscoring THs' critical role in embryogenesis.
