T-2 toxin-induced apoptosis in rat keratinocyte primary cultures

T-2 toxin, a kind of trichothecene mycotoxins produced by the genus Fusarium, induces apoptosis in basal keratinocytes when topically applied to the dorsal skin of rats. In the present study, direct effects of T-2 toxin on keratinocyte primary cultures obtained from newborn rats were examined after the third passage. Keratinocyte medium containing 0.25 microg/ml of T-2 toxin dissolved in dimethyl sulfoxide or solvent alone was added to 4-day cultures and incubated at 37 degrees C. At 0.5, 1, 3, 5, 7, and 9 h after treatment (h), feeder layer was separated from flasks, and cells were trypsinized. Cell viability was estimated by trypan blue exclusion method. In addition, RNA was obtained and RT-PCR was performed. Samples obtained from slide cultures at 3, 6, 9, and 12 h were fixed in 4% paraformaldehyde or 2.5% glutaraldehyde for morphological examination. After T-2 toxin application, cell viability decreased to 40% at 3 h. At 6 h, small-sized keratinocytes showed pyknosis or karyorrhexis, resulting in detachment from slides. The number of such cells increased until 12 h. These small-sized keratinocytes showed ultrastructural changes characteristic for apoptosis. At the same time, large squamous keratinocytes showed intracytoplasmic edema. The expression of apoptosis-related genes (c-fos and c-jun) and cytokines (TNF-alpha and IL-1beta) mRNAs markedly increased before the development of apoptosis. These findings indicate that c-fos and c-jun oncogenes and TNF-alpha and IL-1beta play an important role in the development of T-2 toxin-induced apoptosis in keratinocytes.