Cadmium Induces Apoptosis in the Human Osteoblast-like Cell Line Saos-2

Abstract

Human exposure to the heavy metal cadmium has been associated with the development of bone diseases, including osteoporosis and osteomalacia. The mechanisms by which cadmium exerts a direct effect on bone remain unclear. Bone cells go through apoptosis for proper bone remodeling; therefore, it was hypothesized that cadmium disrupts this normal balance by inducing apoptosis. Human osteoblast-like cells (Saos-2) were treated with 10–200 μM cadmium chloride (CdCl₂) and evaluated by trypan blue staining and phase-contrast microscopy. Exposure to CdCl₂ resulted in decreased cell viability and changes in cell morphology characteristic of apoptosis. The role of apoptosis in cadmium-induced toxicity was further evaluated using the fluorescent marker annexin V, which detects externalization of cell membrane phosphatidylserine. Nuclear changes associated with apoptosis were assessed by Hoechst staining and a DNA fragmentation assay. A significant increase in annexin V-positive cells was observed following CdCl₂ treatment. Nuclear changes associated with apoptosis, including marginalization and condensing of chromatin and DNA fragmentation, were also observed following CdCl₂ treatment. Cadmium-induced apoptosis in Saos-2 cells was also accompanied by an increase in caspase-3 activity. The addition of the caspase-3 inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) or the known cadmium chelating agent potassium bis(2-hydroxyethy)dithiocarbamate, (K[bhedtc]), blocked caspase-3 activation induced by cadmium. Collectively, this study has identified a role for apoptosis in cadmium-induced toxicity in bone cells, and provides insight for future studies on mechanisms underlying the disruption of apoptotic signaling cascades in bone and the relationship to bone disease.

This work was supported by National Institutes of Health grant P20RR016454 from the INBRE Program of the National Center for Research Resources and Sigma Xi Scientific Research Society. The authors thank Dr. Amy Bryant and Clifford Bayer at the Veteran's Affairs Medical Center, Boise, ID, for use of the flow cytometer. The authors also appreciate the helpful advice from Dr. John R. D. Stalvey and Wendy Harvey, and the generous donation of K[bhedtc] from Dr. Peter Craig.