Estimation of increased concentration of intracellular Cd(2+) by fluo-3 in rat thymocytes exposed to CdCl(2).

Cadmium, an environmental pollutant, has been reported to induce apoptosis in murine lymphocytes. To reveal the mechanism of cadmium-induced apoptosis, one of important questions is whether cadmium increases intracellular concentration of Ca(2+) (Ca(2+)), Cd(2+) (Cd(2+)) or both. It is difficult to detect the increase in Ca(2+) using Ca(2+)-chelator-based fluorescent Ca(2+) indicators in the presence of Cd(2+) because of their sensitivity to Cd(2+). Therefore, the study on membrane response such as Ca(2+)-dependent hyperpolarization gives a clue to reveal whether the Ca(2+) or Cd(2+) is increased. Cadmium at concentrations of 3 μM or more dose-dependently augmented fluo-3 fluorescence in rat thymocytes, presumably suggesting an increased Ca(2+). However, the membranes were not hyperpolarized although the cells possess Ca(2+)-dependent K(+) channels. One may argue that cadmium inhibits Ca(2+)-dependent K(+) channels so that cadmium fails to hyperpolarize the membranes. It is unlikely because the Ca(2+) increased by A23187, a calcium ionophore, elicited the hyperpolarization in the presence of Cd(2+). Furthermore, the profile of cytotoxicity induced by cadmium, examined by ethidium bromide and annexin V-FITC, was different from that induced by A23187. Taken together, it is concluded that the application of cadmium increases the Cd(2+) rather than the Ca(2+) in rat thymocytes, resulting in the induction of cytotoxicity.