Biol. Pharm. Bull. 29(4) 648—654 (2006)

morphological and biochemical changes such as cell shrinkage, membrane blebbing, phosphatidylserine (PS) externalization, the potential loss of the mitochondrial membrane, poly (ADP-ribose) polymerase (PARP) cleavage, chromatin condensation and genomic DNA fragmentation. External stimulation may induce apoptosis as well as necrosis. These two types of cell death can be distinguished from each other by using various methods, such as the flow cytometric assay using annexin V and propidium iodide (PI). Annexin V is a calcium-dependent phospholipid-binding protein that possesses a strong affinity with PS, which is externalized onto the cell surface in the early stages of apoptotic cell death. Apoptosis constitutes a crucial process for eliminating cancer cells; antitumor agents with different modes of action have been reported to trigger apoptosis in chemosensitive cells. In many systems, apoptosis is associated with the loss of mitochondrial inner membrane potential (DY m), which may be regarded as a limiting factor in the apoptotic pathway. This loss was reported to be associated with the induction of mitochondrial permeability transition and/or channel-forming by the Bax. Another characteristic of apoptosis is the cleavage of PARP by activated caspases. Activated caspase-3 and/or caspase-7 cleaves PARP to an 89-kDa signature fragment, in a DEVD↓G sequence separating the amino-terminal DNA-binding domain from the carboxyl-terminal catalytic domain of the enzyme. PARP is a nuclear DNA-binding protein of 113 kDa that is constitutively expressed in eukaryotes and that comprises up to 1% of the total nuclear proteins. At lower levels of DNA damage, the PARP inhibits the proapoptotic Ca /Mg endonuclease, and thus protects cells against apoptosis. DNA fragmentation associated with apoptosis is induced by the DNA fragmentation factor (DFF), which is activated by caspases, mainly caspase-3. DFF is composed of two protein subunits, a 40-kDa caspaseactivated nuclease (DFF40/CAD), and its 45-kDa inhibitor (DFF45/ICAD). The cleavage of DFF45/ICAD by caspase-3 releases DFF40/CAD from its inhibitor, leading to the induction of nuclease activity, nuclear condensation, and DNA fragmentation in vitro. TUNEL (TdT-mediated dUTP nickend labeling) assay usually involves the terminal nucleotide transferase-mediated polymerization of labelled dUDP, in a template-independent manner, at the site of DNA breaks at 3 -OH. Mycotoxins of scirpenol belong to the trichothecene family, which is produced mainly by the fungal species of the genus Fusarium. The chemical structure of scirpenol has three hydroxyl groups which can be acetoxylated during the biosynthesis in Fusarium. Further metabolized, fully acetoxylated scirpenol becomes T-2 toxin, which is known as a highly toxic mycotoxin. Acetoxylated scirpenol also has very high toxicities in animas and humans. The cytotoxic compound of 4b-acetoxyscirpendiol (4-MAS) is a C-4 monoacetoxylated form of scirpenol, a subfamily of trichothecene mycotoxins. Trichothecenes were reported to inhibit protein synthesis by binding to the ribosomal peptidyl transferase site. 4-MAS isolated from Isaria japonica induced apoptosis though caspase-3 activation and DNA frag648 Vol. 29, No. 4 Biol. Pharm. Bull. 29(4) 648—654 (2006)