Exposure to cadmium poses a threat to human health, including increased susceptibility to developing the bone disease osteoporosis. Despite its recognized importance as an environmental toxin, little is known about how cadmium directly impacts bone-forming osteoblasts. We previously reported that cadmium induces apoptosis in human osteoblast-like Saos-2 cells. In this work, we hypothesize that cadmium exposure induces oxidative stress which leads to decreased RUNX2 mRNA expression and increased apoptotic death, and predict that the antioxidant NAC mitigates the damaging effects of cadmium. Oxidative stress is implicated in osteoporosis; furthermore the osteoblast transcriptional factor RUNX2 is reported to play a protective role against osteoporosis in postmenopausal women. Cells treated with 10 μM CdCl 2 exhibited signs of oxidative damage including depletion in glutathione, increased reactive oxygen species formation, and enhanced lipid peroxidation. RUNX2 mRNA expression, by RT-PCR, was significantly reduced after exposure to 10 μM CdCl 2 . Pretreatment with the antioxidant NAC (1 mM) prevented cadmium-induced decrease in RUNX2 mRNA and protected cells from apoptotic death. This study provides insight into the mechanisms underlying cadmium-induced osteotoxicity. In addition, this study distinguishes itself by identifying RUNX2 as a target for heavy metal-induced osteotoxicity.

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 muM 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.

Electronic cigarettes (e‐cigarettes) are nicotine delivery devices advertised as a healthier alternative to conventional tobacco products, but their rapid rise in popularity outpaces research on potential health consequences. As conventional tobacco use is a risk factor for osteoporosis, this study examines whether exposure to electronic liquid (e‐liquid) used in e‐cigarettes affects bone‐forming osteoblasts. Human MG‐63 and Saos‐2 osteoblast‐like cells were treated for 48 hours with 0.004%‐4.0% dilutions of commercially available e‐liquids of various flavors with or without nicotine. Changes in cell viability and key osteoblast markers, runt‐related transcription factor 2 and Col1a1, were assessed. With all e‐liquids tested, cell viability decreased in a dose‐dependent manner, which was least pronounced in flavorless e‐liquids, most pronounced in cinnamon‐flavored e‐liquids and occurred independently of nicotine. Col1a1, but not runt‐related transcription factor 2, mRNA expression was upregulated in response to coffee‐flavored and fruit‐flavored e‐liquids. Cells treated with a non‐cytotoxic concentration of fruit‐flavored Mango Blast e‐liquid with or without nicotine showed significantly increased collagen type I protein expression compared to culture medium only. We conclude that the degree of osteotoxicity is flavor‐dependent and occurs independently of nicotine and that flavored e‐liquids reveal collagen type I as a potential target in osteoblasts. This study elucidates potential consequences of e‐cigarette use in bone.

Recent reports of cadmium in electronic waste and jewelry have increased public awareness regarding this toxic metal. Human exposure to cadmium is associated with the development of osteoporosis. We previously reported cadmium induces apoptosis in human tumor-derived Saos-2 osteoblasts. In this study, we examine the extracellular signal-regulated protein kinase (ERK) and protein kinase C (PKC) pathways in cadmium-induced apoptosis and altered osteoblast gene expression. Saos-2 osteoblasts were cultured in the presence or absence of 10 μM CdCl2 for 2–72 hours. We detected significant ERK activation in response to CdCl2 and pretreatment with the ERK inhibitor PD98059 attenuated cadmium-induced apoptosis. However, PKCα activation was not observed after exposure to CdCl2 and pretreatment with the PKC inhibitor, Calphostin C, was unable to rescue cells from cadmium-induced apoptosis. Gene expression studies were conducted using qPCR. Cells exposed to CdCl2 exhibited a significant decrease in the bone-forming genes osteopontin (OPN) and alkaline phosphatase (ALP) mRNA. In contrast, SOST, whose protein product inhibits bone formation, significantly increased in response to CdCl2. Pretreatment with PD98059 had a recovery effect on cadmium-induced changes in gene expression. This research demonstrates cadmium can directly inhibit osteoblasts via ERK signaling pathway and identifies SOST as a target for cadmium-induced osteotoxicity.

The heavy metal cadmium is a widespread environmental contaminant that has gained public attention due to the global increase in cadmium-containing electronic waste. Human exposure to cadmium is linked to the pathogenesis of osteoporosis. We previously reported cadmium induces apoptosis and decreases alkaline phosphatase mRNA expression via extracellular signal-regulated protein kinase (ERK) activation in Saos-2 bone-forming osteoblasts. This study examines the mechanisms of cadmium-induced osteotoxicity by investigating roles of Ca+2/calmodulin-dependent protein kinase (CAMK) pathways. Saos-2 or MG-63 cells were treated for 24 or 48 h with 5 μM CdCl2 alone or in combination with calmodulin-dependent phosphodiesterase (PDE) inhibitor CGS-9343β; calmodulin-dependent kinase kinase (CAMKK) inhibitor STO-609; or calmodulin-dependent kinase II (CAMKII) inhibitor KN-93. CGS-9343β protected against cadmium-induced toxicity and attenuated ERK activation; STO-609 enhanced toxicity and exacerbated ERK activation, whereas KN-93 had no detectable effect on cadmium-induced toxicity. Furthermore, CGS-9343β co-treatment attenuated cadmium-induced apoptosis; but CGS-9343β did not recover cadmium-induced decrease in ALP activity. The major findings suggest the calmodulin-dependent PDE pathway facilitates cadmium-induced ERK activation leading to apoptosis, whereas the CAMKK pathway plays a protective role against cadmium-induced osteotoxicity via ERK signaling. This research distinguishes itself by identifying pleiotropic roles for CAMK pathways in mediating cadmium’s toxicity in osteoblasts.