Cytotoxic and apoptotic effects of cobalt and chromium ions on J774 macrophages - Implication of caspase-3 in the apoptotic pathway

The aim of this study was to evaluate the cytotoxic and apoptotic effects o f cobalt and chromium ions on macrophages in vitro, and analyze the implica tion of caspase-3 in the apoptotic pathway. J774 mouse macrophages (5 x 10( 5) cells/ml) were exposed for up to 24 h to 0-10 ppm Co2+ and 0-500 ppm Cr3 +. The cytotoxic effect of ions was measured by Trypan blue exclusion. DNA analysis on agarose gel was used as a specific test for detection of DNA fr agmentation into oligonucleosomes that occurs in apoptotic cells. The prote olytic cleavage of poly(ADP-ribose)polymerase (PARP), closely associated wi th the induction of apoptosis, was also analyzed along with the appearance of the active fragment of caspase-3, implicated in several apoptosis pathwa ys. Results demonstrated that both Co2+ and Cr3+ ions induce macrophage mor tality in a dose-dependent manner. However, Co2+ is more toxic inducing a c ell mortality up to 28% with only 10 ppm vs. 37% with 500 ppm of Cr3+. DNA analysis demonstrated that both Co2+ and Cr3+ ions induce DNA fragmentation , between 6-10 ppm Co2+ and 250-500 ppm Cr3+ after 24 h incubation. PARP cl eavage and the appearance of caspase-3 active fragment were observed after 6 h with both Co+ and Cr3+ ions, with a stronger signal after 24 h and 10 p pm of Co2+ or 500 ppm of Cr3+. In conclusion, this study demonstrates that after 24 h incubation, both Co2+ and Cr3+ ions can induce macrophage mortal ity, and more specifically apoptosis. The results also suggest that apoptos is occurs via a caspase-3 pathway. However, the relative importance of necr osis and apoptosis and the effects of longer exposure times on the inductio n of macrophage death by these metal ions remain to be investigated. (C) 20 01 Kluwer Academic Publishers.