Uncovering Thyroid Vulnerability to Doxorubicin: Integrative Cellular and in vivo Evidence of Mitochondrial Dysfunction.

Objective: Doxorubicin (DOX), a first-line anthracycline chemotherapeutic for triple-negative breast cancer (TNBC), is known to cause severe off-target toxicities including cardiotoxicity. However, its effects on the thyroid, a key regulator of systemic metabolism and long-term health, have been largely overlooked. Our study addresses this gap by investigating whether DOX directly impairs thyroid integrity and elucidating the underlying mechanisms. Materials and Methods: An integrated experimental strategy was employed, combining primary mouse thyroid follicular epithelial (MTFE) cells, zebrafish (AB strain) larvae, and multidisciplinary molecular and histopathological approaches. To better simulate the physiological context of chemotherapy, MTFE cells were exposed to DOX that had been effluxed from TNBC cells. Oxidative stress, mitochondrial function, and apoptotic activity were assessed in MTFE cells, while zebrafish larvae were treated with DOX to evaluate thyroid hormone secretion, inflammatory cytokine levels, and fibrotic changes. Key biomarkers were analyzed via immunofluorescence and histological staining. Results: In MTFE cells, DOX triggered significant oxidative stress, reflected by elevated malondialdehyde (MDA) levels, and led to mitochondrial dysfunction, evidenced by ultrastructural abnormalities and loss of JC-1 membrane potential. In zebrafish, DOX exposure resulted in an approximately 20% reduction in T3/T4 levels, accompanied by a 1.4-fold increase in TNF-α, indicating that the hormone shift serves as a marker of inflammatory thyroid injury. These findings establish a direct link between DOX-induced oxidative and mitochondrial damage and the structural and functional deterioration of thyroid tissue. Conclusion: Our findings reveal a previously underappreciated thyrotoxic effect of DOX, primarily mediated via oxidative stress-driven mitochondrial dysfunction and apoptotic signaling. These insights underscore the novelty of the thyroid as a vulnerable target during DOX-based chemotherapy and emphasize the need for routine thyroid monitoring in clinical practice. This work further supports developing adjunct interventions to mitigate thyroid damage and improve long-term safety in cancer survivors.