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

Abstract

The aim of this study was to evaluate the cytotoxic and apoptotic effects of cobalt and chromium ions on macrophages in vitro, and analyze the implication of caspase-3 in the apoptotic pathway. J774 mouse macrophages (5×10⁵ 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 fragmentation into oligonucleosomes that occurs in apoptotic cells. The proteolytic cleavage of poly(ADP-ribose)polymerase (PARP), closely associated with the induction of apoptosis, was also analyzed along with the appearance of the active fragment of caspase-3, implicated in several apoptosis pathways. Results demonstrated that both Co²⁺ and Cr³⁺ ions induce macrophage mortality in a dose-dependent manner. However, Co²⁺ is more toxic inducing a cell 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 cleavage 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 ppm 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 mortality, and more specifically apoptosis. The results also suggest that apoptosis occurs via a caspase-3 pathway. However, the relative importance of necrosis and apoptosis and the effects of longer exposure times on the induction of macrophage death by these metal ions remain to be investigated.

© 2001 Kluwer Academic Publishers