Gliotoxin induces apoptosis in cultured macrophages via production of reactive oxygen species and cytochrome c release without mitochondrial depolarization

The cytotoxicity and its underlying mechanisms induced by gliotoxin (GT), a n immunosuppressive agent, in macrophages are poorly understood. We report here that GT induced a rapid apoptosis (DNA fragmentation and hypodiploid n uclei obtained within 4hrs of treatment) in murine macrophages PU5-1.8 in a dose-dependent and cell cycle-independent manner. The GT-induced apoptosis was suppressed by z-Asp, z-VAD-fmk and antioxidants suggesting that produc tion of reactive oxygen species (ROS) and activation of caspases were impor tant in this process. Also, release of cytochrome c from mitochondria was f ound to be an early event (within I hr) after addition of GT (250 ng/ml) an d its presence in the cytosol was sufficient to elicit apoptosis. Interesti ngly, the release of cytochrome c was not accompanied by a reduction in the mitochondrial membrane potential (psi (m)) as determined by several psi (m )-sensitive fluorescent indicators. Taken together, our results indicate th at GT is a potent apoptotic agent in PU5-1.8 cells and the loss of psi (m) is not a universal early marker for apoptosis.