Hydrogen peroxide-induced cytotoxicity in cultured epithelial cells (WISH): A functional and morphological study

The effect of hydrogen peroxide on cultured epithelial cells (WISH) was investigated with particular emphasis on cell functions: cell morphology and cytoskeletal components were also studied. The presence of low concentrations of H(2)O(2) (0.1-0.4 mM) in the culture medium markedly inhibited cell growth, although WISH contained catalase and glutathione-peroxidase activities. After 1 hr of incubation with H(2)O(2) up to 5 mM, the majority of the cells were still alive, but reincubation in normal medium for 24 hr clearly reduced cell viability. Cell adhesion was dose-dependently reduced by H(2)O(2) treatment (0.1-0.5 mM) for 4 hr. Incubation with 1.5 mM-H(2)O(2) gave rise to a bleb appearance on the cell surface and to mitochondrial swelling, as shown, respectively, by scanning and transmission electron microscopy. Immunofluorescence studies revealed changes in microtubules and microfilaments, which are two of the main cytoskeletal components. The modification of microtubules was also confirmed by Western blot analysis of WISH protein homogenates submitted to SDS-PAGE. WISH treated with 1.5 mM-H(2)O(2) showed decreased levels of GSH compared with control cells: glutathione transferase activity was reduced, whereas other enzymes of the glutathione cycle were unchanged.