Electronic cigarette liquid exposure induces flavor-dependent osteotoxicity and increases expression of a key bone marker, collagen type I.

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.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases human hepatic stellate cell activation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a halogenated aromatic hydrocarbon that elicits toxicity through the aryl hydrocarbon receptor (AhR). In the liver, gross markers of TCDD toxicity are attributed to AhR activation in parenchymal hepatocytes. However, less is known regarding the consequences of TCDD treatment on non-parenchymal cells in the liver. Hepatic stellate cells (HSCs) are non-parenchymal cells that store vitamin A when quiescent. Upon liver injury, activated HSCs lose this storage ability and instead function in the development and maintenance of inflammation and fibrosis through the production of pro-inflammatory mediators and collagen type I. Reports that TCDD exposure disrupts hepatic retinoid homeostasis and dysregulates extracellular matrix remodeling in the liver led us to speculate that TCDD treatment may disrupt HSC activity. The human HSC line LX-2 was used to test the hypothesis that TCDD treatment directly activates HSCs. Results indicate that exposure to 10nM TCDD almost completely inhibited lipid droplet storage in LX-2 cells cultured with retinol and palmitic acid. TCDD treatment also increased LX-2 cell proliferation, expression of α-smooth muscle actin, and production of monocyte chemoattractant protein-1 (MCP-1), all of which are characteristics of activated HSCs. However, TCDD treatment had no effect on Col1a1 mRNA levels in LX-2 cells stimulated with the potent profibrogenic mediator, transforming growth factor-ß. The TCDD-mediated increase in LX-2 cell proliferation, but not MCP-1 production, was abolished when phosphoinositide 3-kinase was inhibited. These results indicate that HSCs are susceptible to direct modulation by TCDD and that TCDD likely increases HSC activation through a multi-faceted mechanism.

Cadmium exposure activates the ERK signaling pathway leading to altered osteoblast gene expression and apoptotic death in Saos-2 cells.

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 10µM CdCl(2) for 2-72h. We detected significant ERK activation in response to CdCl(2) and pretreatment with the ERK inhibitor PD98059 attenuated cadmium-induced apoptosis. However, PKCα activation was not observed after exposure to CdCl(2) 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 CdCl(2) 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 CdCl(2). 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.

Cadmium-induced decrease in RUNX2 mRNA expression and recovery by the antioxidant N-acetylcysteine (NAC) in the human osteoblast-like cell line, Saos-2.

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 microM CdCl2 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 microM CdCl2. Pretreatment with the antioxidant NAC (1mM) 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.

Characterization and in vitro cytotoxicity testing of ethanolamine-derived cadmium chelating agents.

We have synthesized and characterized the new cadmium chelating agent potassium bis(2-hydroxyethyl)aminoethyldithiocarbonate hemihydrate, K[bhexan] x 0.5H2O (2), that is structurally related to the known effective in vivo cadmium chelating agent potassium bis(2-hydroxyethyl)dithiocarbamate, K[bhedtc] (1). The corresponding cadmium complex of 2 differs from di(bis(2-hydroxyethyl)dithiocarbamato)cadmium(II), Cd(bhedtc)2 (3), in that the insoluble compound exhibits an elemental composition consistent with a cadmium:ligand ratio of 2:1. The cytotoxicity of the 1-3 was investigated using the human osteoblast-like cell line, Saos-2. Compounds 1 or 2 did not affect cell adherence or cell viability in the 100-500 microM concentration range studied, whereas 3 resulted in a concentration-dependent increase in loss of cell adherence and decrease in cell viability. Overall, the results of the loss of cell adherence, trypan blue exclusion and MTT assays showed that administration of 3 (cadmium complex of 1) resulted in cytotoxicity lower than that of cadmium chloride, but higher than that of the chelator 1 alone. The effect of simultaneous addition of cadmium chloride and 1 or 2 on cell viability was also assessed using the MTT assay. For the 100 microM cadmium chloride experiments, cell viability comparable to control cells was achieved for both 1 and 2 in the 100-500 microM concentration range studied. Cell viability comparable to control cells was achieved for 1 but not 2 in the 100-500 microM concentration range studied for the 200 microM cadmium chloride experiments. Thus 1 appears more effective than 2 in the ability to mediate the cytotoxic effects of cadmium in vitro upon concomitant administration.