Characterization of cadmium-induced apoptosis in rat lung epithelial cells: evidence for the participation of oxidant stress

The mode of cadmium-induced cell death was investigated in a rat lung epith elial cell line. Cells, grown to near confluence, were exposed to 0-30 mu M CdCl2 for 0-72 h. Phase contrast microscopy and fluorescent nuclear staini ng showed that Cd caused morphological alterations in lung epithelial cells that are characteristic of apoptosis. These changes included cell shrinkag e, detachment of the cell from its neighbors, cytoplasmic and chromatin con densation, and fragmentation of the nucleus into multiple chromatin bodies surrounded by remnants of the nuclear envelope. Apoptotic DNA degradation w as validated and quantitated using a sensitive enzyme-linked immunosorbent assay (ELISA) which measures the amount of histone-bound DNA fragments in t he cytosol. Using this technique, a maximum level of apoptosis (5-fold high er than control) was observed in cultures exposed for 48 h to 20 mu M CdCl2 . The terminal deoxyribonucleotidyl transferase mediated dUTP nick end labe ling method (TUNEL) was subsequently used to determine the percentage of ce lls that contained Cd-induced DNA strand breaks. After 48 h, approximately 54% of the cells exposed to 20 mu M Cd were TUNEL positive compared to less than 2% for control cells. Although the mechanisms by which Cd initiates a poptosis in these cells are presently not known, reactive oxygen species ar e likely to play a role. This possibility is supported by the finding that the first morphological features indicative of apoptosis were preceded by t he up-regulation of oxidant stress genes (glutathione S-transferase-alpha, gamma-glutamylcysteine synthetase, and metallothionein-1), activation of re dox sensitive transcription factors (AP-1 and NF-kappa-B), and changes in v arious forms of glutathione (reduced, oxidized, and protein-bound). (C) 199 9 Elsevier Science Ireland Ltd. All rights reserved.