Fluquinconazole disrupts calcium and mitochondrial homeostasis, leading to bovine mammary epithelial cell death

Pesticides are essential for improving productivity, but their residues in animal feed can affect livestock, food produced by livestock, and, ultimately, humans. Fluquinconazole is a widely used triazole fungicide. To date, there are no detailed reports on the mechanisms underlying fluquinconazole toxicity in cells. In the present study, we explored the toxic effects of fluquinconazole on bovine mammary epithelial (MAC-T) cells. Fluquinconazole significantly reduced the cell viability in both two-dimensional and three-dimensional cultures of cells. Additionally, cell cycle analysis showed an increase in the number of G2/M phase cells, demonstrating the occurrence of cell cycle arrest. Calcium is an important mediator of various cellular signaling pathways. After fluquinconazole treatment, the concentration of calcium ions in the cytosol decreased, whereas that in the mitochondria increased. This imbalance damages mitochondrial membrane potential and reduces respiratory efficiency, ultimately causing mitochondrial dysfunction. Fluquinconazole modified the phosphorylation levels of MAPK/PI3K pathway proteins compared to those in the control group. Furthermore, endoplasmic reticulum (ER) stress and mitochondria-associated ER membrane protein expression were increased. These changes led to an elevation in the expression of pro-apoptotic proteins and a decline in the expression of anti-apoptotic proteins, thereby activating the apoptotic pathway in MAC-T cells. In conclusion, fluquinconazole induced mitochondrial dysfunction and ER stress, resulting in the death of MAC-T cells.