| Summary: | 博士 === 國立清華大學 === 生命科學系 === 94 === Cadmium (Cd) compounds are ubiquitous environmental contaminants that have been classified as human carcinogens. However, the toxicological mechanisms of Cd are not well understood. In this study, we investigated the cytotoxicity of Cd toward Chinese hamster ovary (CHO) K1 cells. Analysis of cell growth using cell counting and BrdU incorporation indicated that there was no difference in growth rate when less than 0.4 μM Cd was given within 24 h. A dose-dependent reduction of cell proliferation was observed when more than 0.4 μM Cd was added. Flow cytometric analysis indicated that cells were arrested at G2/M phase when 0.8 to 2 μM Cd was given for 24 h. However, necrosis were induced when more than 2 μM Cd was administered. This effect can be efficiently inhibited by a programmed necrosis inhibitor, necrostatin-1 (Nec-1). Despite the fact that Cd treatment arrested cells mainly in the G2 but not M phase, an elevation in the cyclin-dependent kinase 1 (Cdk1) activity was found. Analysis of intracellular Cd content showed that only cells received 7.5 to 17.5 ng/106 cells progressed and retarded at G2/M phase. When synchronized cells at G1/S border were treated with 1 μM Cd, cells were delayed at the S phase progression and then arrested at G2/M phase; When synchronized G2 cells were treated, cells passed through G2/M and G1 phase, and then accumulated at S phase.
Since Cd stimulated the production of reactive oxygen species (ROS) and leads to cell damage, we investigated the involvement of ROS with necrosis. Intracellular ROS level increased after treatment with 1 μM for 24 h or 4 μM for 8 h. Antioxidants (BHA) that effectively reduced ROS production did not alter the G2/M arrest but inhibit necrosis after Cd treatment. This finding suggests that the Cd-induced ROS leads to cell necrosis but not G2/M arrest. Interestingly, we found that EUK-8, a SOD/catalase mimics, could rescue cells from G2/M arrest and necrosis. Analysis of intracellular Cd content indicated that EUK-8 reduced cellular Cd accumulation via blockage of Cd uptake into cells rather than promotion of Cd release from cells. EUK-8 is a Mn-salen complex. Mn decreased the uptake and cytotoxicity of Cd, while salen perturbed the membrane integrity and increased the uptake and cytotoxicity of Cd. Electronic paramagnetic resonance (EPR) spectrophotometric analysis indicated that only 0.09% of free manganese (Mn2+) dissolved in EUK-8 solution, which did not inhibit Cd-induced G2/M arrest. This finding indicates that it is EUK-8 itself that reduces Cd toxicity.
Because Cd-induced necrosis was a predominantly toxic effect in CHO cells, we further studied the mechanisms involved. Analysis of intracellular calcium (Ca2+) level indicated that 4 μM Cd caused Ca2+-overload. Intracellular Ca2+ chelator (BAPTA-AM) completely inhibited Cd-induced Ca2+-overload and necrosis. These findings suggests that Ca2+-overload played the most dominant role on Cd-induced necrosis. Calpain is a Ca2+-dependent protease. Analysis of calpain activity indicated that Cd activated calpain. Treatment with calpain inhibitors (calpeptin) or overexpression of endogenous calpain inhibitors (calpastatin) partially inhibited Cd-induced necrosis. BAPTA-AM but not calpeptin reduced Cd-induced ROS, suggesting that calpain activation and ROS production are two independent downstream signalings of Cd-induced Ca2+-overload. It has been known that mitochondrial membrane permeability (MPT) is critical for necrosis induction. Induction of MPT causes collapse of mitochondrial membrane potential (MMP). The MMP level dropped when cells received Cd treatment. MPT inhibitor (cyclospron A, CsA) rescued cells from MMP reduction and necrosis. Cd-induced MPT was also inhibited by BAPTA-AM and calpeptin, which indicating that alternation of MPT is a downstream signal of calpain. NF-κB is a transcription factor that promotes cell survival. Reporter gene analysis indicated that Cd reduced basal NK-κB activity, which was recovered by BAPTA-AM and BHA. Thus, Cd induced necrosis through calpain-triggered MPT and ROS-dependent basal NF-κB inhibition. We also showed in this study that Nec-1 protected cells from Cd-induced necrosis via inhibition of calpain/MPT pathway and activation of NF-κB activity.
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