Oxyfluorfen induces cell cycle arrest by regulating MAPK, PI3K and autophagy in ruminant immortalized mammary epithelial cells

Oxyfluorfen, a phenoxy phenyl-type herbicide, causes significant damage to ecosystems through chronically effecting invertebrates, fish, and mammals. Considering its adverse effect on ecosystem conservation, it is necessary to investigate its toxic effects on animals. However, the mechanisms of oxyfluorfen toxicity on bovines are not well established. This study investigated the cytotoxic effect of oxyfluorfen on bovine mammary epithelial cells (MAC-T). We conducted several functional experiments to examine the response of MAC-T to oxyfluorfen under various concentrations (0, 1, 2, 5, and 10 ppm). Oxyfluorfen decreased cell viability and increased apoptotic cells by regulating the expression of apoptotic genes and proteins in MAC-T. In addition, oxyfluorfen-treated cells exhibited reduced PCNA expression with a low 3D spheroid formation as compared to that of control cells. Furthermore, oxyfluorfen treatment suppressed cell cycle progression with a decrease in cyclin D1 and cyclin A2 in MAC-T. Next, we performed western blot analysis to verify intercellular signaling changes in oxyfluorfen-treated MAC-T. The phosphor-AKT protein was increased, whereas MAPK signal pathways were decreased. Particularly, the combination of oxyfluorfen with U0126 or SP600125 completely blocked the ERK1/2 and JNK pathways leading to cell viability in MAC-T. Moreover, oxyfluorfen induced inflammatory gene expression and autophagy by increasing phosphorylation of P62 and LC3B in MAC-T. These results demonstrated that oxyfluorfen has cytotoxic effect on MAC-T, implying that the milk production capacity in cows may eventually harm humans.