Phenethyl isothiocyanate induces IPEC-J2 cells cytotoxicity and apoptosis via S-G₂/M phase arrest and mitochondria-mediated Bax/Bcl-2 pathway

Phenethyl isothiocyanate (PEITC) is one of the glucosinolates (GLs) present in cruciferous vegetables. Although there are many reports of livestock and poultry poisoning caused by plants containing GLs, the actual dosage that causes poisoning and the characteristics of GLs and their metabolites are unclear. Herein, we investigated the inhibitory effects of PEITC on IPEC-J2 cells and examined the mechanisms of PEITC-induced apoptosis via the mitochondrial pathway. Cell viability was determined by the MTT assay, and the levels of reactive oxygen species, mitochondrial membrane potential (∆Ψ), intracellular Ca²⁺ concentration, and cell apoptosis were detected by flow cytometry. IPEC-J2 cells were collected to assess the activities of superoxide dismutase, catalase, and glutathione peroxidase, as well as the contents of glutathione, malondialdehyde, H₂O₂, ATP, and lactate dehydrogenase, using biochemical methods. The levels of cytochrome c, Bax, Bcl-2, caspase-3, caspase-9, poly (ADP-ribose) polymerase (PARP)-1, p53, CDC25C, and cyclin A2 were analyzed by western blotting. We found that PEITC effectively inhibited the growth of IPEC-J2 cells, causing apoptosis. PEITC suppressed the level of mitochondrial membrane potential; released cytochrome c from the mitochondria to the cytoplasm; reduced ATP levels; inhibited Bcl-2 expression; increased Bax expression; and activated caspase-9, caspase-3, and PARP-1, leading to apoptosis. PEITC also induced G₂/M and S phase arrest by affecting cell cycle-associated proteins such as p53, CDC25C, and cyclin A2. We conclude that PEITC causes oxidative stress, cell cycle arrest, and apoptosis in IPEC-J2 cells via a mitochondrial-dependent Bax/Bcl-2 pathway.