Abstract
T-2 Toxin (12-13 epoxytrichothecene mycotoxin) induces apoptosis in basal keratinocytes when topically applied to the dorsal skin of Wistar-derived hypotrichotic WBN/ILA-Ht rats. In the present study, the effects of T-2 toxin on rat keratinocyte primary cultures were examined. Keratinocytes which were obtained from newborn Wistar rats and cultured by the method of Reynwald and Green (14) were used after the third passage. Keratinocyte medium containing 0.25μg/ml of T-2 toxin dissolved in DMSO or solvent alone was added to four-days-cultures and incubated at 37°C. At 0.5, 1, 3, 5, 7, and 9 hours after treatment (hr), feeder layer was separated from T-2 toxin-treated and control flasks, and cells were trypsinized. Cell viability was estimated by trypan blue exclusion method. In addition, RNA was obtained and RT-PCR was perfomed. Samples obtained from slide cultures at 3, 6, 9 and 12hr were fixed in 4% paraformaldehide or 2.5% glutaraldehide for morphological examination. After T-2 toxin application, cell viability decreased to 40% at 12hr. Pyknotic or karyorrhectic nuclei with cell body shrinkage were found in small sized keratinocytes at 6 hr, and their number increased until 12hr. These small sized keratinocytes showed ultraestructural changes characteristic for apoptosis; condensation of nuclear chromatine and phagocytosis of apoptotic bodies by neibour keratinocytes. At the same points, large squamous keratinocytes showed degeneration characterized by intracytoplasmic edema. The expression of apoptosis-related genes (c-fos and c-jun) and cytokines mRNA, TNF-α and IL-1β markedly increased prior to the development of apoptosis. These findings suggest that c-fos and c-jun oncogenes and TNF-α and IL-1β may play an important role in the development of T-2 toxin-induced apoptosis in basal keratinocytes.
