Diflubenzuron leads to apoptotic cell death through ROS generation and mitochondrial dysfunction in bovine mammary...

Pesticides, which are used in agriculture and forestry to eliminate insects, are a major cause of environmental pollution. Among them, diflubenzuron (DFB), 1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl) urea, is a common benzoylurea insecticide that hinders larval development, primarily in Aedes aegypti larvae. Many experts have announced the biological toxicity of DFB in various species. However, the toxicity of benzoylurea pesticides, including DFB, to bovine mammary epithelial cells (MAC-T) is unclear. Therefore, in this study, we confirmed the cytotoxic effects of DFB on the viability and proliferation of MAC-T cells. Additionally, we observed that DFB induced lipid peroxidation through reactive oxygen species (ROS) production, resulting in an increase in transcriptional gene expression related to inflammatory response. Moreover, we demonstrated mitochondrial dysfunction including depolarization of the mitochondrial membrane, perturbation of calcium homeostasis, and, eventually, apoptosis. Furthermore, we identified DFB-triggered signaling pathways related to ROS generation and cell proliferation, as well as their interactions, by treating the cells with pharmacological inhibitors in combination with DFB. DFB attenuated the phosphorylation of AKT, P70S6K, S6, and ERK1/2 and facilitated the phosphorylation of JNK and c-Jun. These results show that DFB can induce apoptotic cell death via ROS generation and mitochondrial dysfunction in MAC-T cells.