Integrated cellular and molecular evidence of Amitraz-induced oxidative stress in SH-SY5Y spheroids

Amitraz (AMZ), a formamidine acaricide widely used in apiculture, is frequently detected in honey at concentrations exceeding regulatory limits; however, its toxicological mechanisms in human systems remain poorly characterized. In this study, a spheroidal SH-SY5Y model was employed to investigate AMZ-induced oxidative stress under conditions that closely mimic structural and functional complexity of the in vivo models. Spheroids were exposed three concentrations of AMZ (IC 50 , IC 50 /2, and IC 50 /3) for 24 h. The levels of cytotoxicity, oxidative stress parameters, and antioxidant responses were evaluated. AMZ exposure resulted in spatially heterogeneous cell death within the spheroid structure and a significant increase in intracellular reactive oxygen species (ROS) at the highest concentration. No significant changes were detected in mitochondrial superoxide production or mitochondrial membrane potential under the experimental conditions tested. At the level of transcription, AMZ (IC 50 /2 and IC 50) induced a selective upregulation of antioxidant and cytoprotective genes, including HMOX1 , NQO1 , and GPX1 . Overall, AMZ induces a spatially heterogeneous oxidative stress response in SH-SY5Y spheroids, characterized by intracellular redox imbalance, without mitochondrial involvement. The findings of this study underscore the significance of employing three-dimensional models to achieve a more profound comprehension of the deleterious effects that pesticides exert on complex organisms, such as spheroids, in contrast to the two-dimensional model that is presently utilized for this purpose.