Elsevier

Toxicology

Volume 308, 7 June 2013, Pages 138-145
Toxicology

Sodium fluoride induces apoptosis in odontoblasts via a JNK-dependent mechanism

https://doi.org/10.1016/j.tox.2013.03.016Get rights and content

Highlights

  • NaF is cytotoxic to odontoblast cells in a dose- and time-dependent manner.

  • Treatment with NaF at 4 mM for 24 h induces apoptosis in OLC cells.

  • Mitochondrial dysfunction is correlated with fluoride-induced apoptosis.

  • JNK signaling mediates mitochondrial dysfunction-dependent NaF-induced apoptosis.

Abstract

Sodium fluoride (NaF) is widely used for the treatment of dental caries and dentin hypersensitivity. However, its pro-apoptotic effect on odontoblasts may lead to harmful side-effects. The purpose of this study was to evaluate the pro-apoptotic effects of NaF in odontoblasts and elucidate the possible underlying molecular mechanisms. NaF generated cytotoxic effects in odontoblast-lineage cell (OLC) in a dose- and time-dependent manner. Exposure of cells to 4 mM NaF for 24 h induced caspase-3 activation, ultrastructural alterations, and resulted in the translocation of Bax to the mitochondria and the release of cytochrome c from the mitochondrial inter-membrane space into the cytosol, indicating that fluoride-mediated apoptosis is mitochondria-dependent. Fluoride treatment also increased phosphorylation of JNK and ERK, but not p38, and apoptosis induced by fluoride was notably or partly suppressed by treatment with JNK or ERK inhibitors, respectively.

Taken together, these findings suggest that NaF induces apoptosis in OLC odontoblasts through a JNK-dependent mitochondrial pathway.

Graphical abstract

Schematic diagram of the signaling pathway involved in NaF-induced apoptosis in OLC cells. In the proposed model, NaF causes apoptosis in OLC cells via activation of the JNK-MAPK pathway, resulting in the induction of signaling cascades responsible for mitochondria-dependent initiation of apoptosis.

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Introduction

Odontoblasts perform a crucial function in dentin formation by producing organic matrix and contributing to mineralization. Odontoblasts deposit new layers of reactionary/reparative dentine and play a role in preventing dentine sensitivity. Apoptosis is a key mechanism by which the life span of odontoblasts is regulated. External stimuli, such as abrasion, dental caries, and cavity preparation induce apoptosis in odontoblasts and promote pulp stem cell migration and differentiation into newly formed odontoblasts (Kitamura et al., 2001, Mitsiadis and De Bari, 2008, Smith, 2002, Toit et al., 2008), resulting in the secretion of reparative dentin. Temperature stimulation and changes in external and internal pressure can also initiate apoptotic events, inducing secondary dentin production (Bronckers et al., 2000, Harada et al., 2008, Kitamura et al., 2003). Decreased numbers of odontoblasts resulting from apoptosis could lead to the reorganization of odontoblasts into a single layer and subsequent secondary dentin deposition (Franquin et al., 1998). Multiple studies have shown that certain toxic substances, such as 2-Hydroxyethyl methacrylate(HEMA), polymethylmethacrylate (PMMA), fluoride and resin monomer in dental materials may also lead to uncontrolled apoptosis in odontoblasts (El-kholany et al., 2011, Hoppe et al., 2011, Krifka et al., 2012, Yamada et al., 2009).

Fluoride is a key ingredient of numerous dental products that are used daily, such as dentifrices used for preventing dentin hypersensitivity and remineralization of dental caries (Burwell et al., 2009, Lee et al., 2010). Currently, many commercial dental products contain high concentrations of fluoride. For example, an in-office product sold under the brand name Gluma® (Heraeus Kulzer, Inc., South Bend, IN, USA) contains fluoride at a concentration of over 1450–4500 ppm (Cummins, 2009). When this type of product is applied to dentin exposed in open tubule orifices or patent tubules, the local concentration of fluoride may reach as high as 5000 ppm (Duane, 2012, Nordstrom and Birkhed, 2010). Concentrations of fluoride at this level may produce significant toxic effects on odontoblasts that cannot be ignored. The cytotoxic effects induced inflammation on odontoblasts (Goldberg et al., 2008) are the link between initial inflammation/apoptosis and cell commitment in the pulp reparative process (Horst et al., 2011).

Abundant evidence has demonstrated the pro-apoptotic effect of NaF in various cell types, including ameloblasts, osteoblasts, neural progenitor cells and odontoblasts (Bronckers et al., 2009, Karube et al., 2009, Kubota et al., 2005, Ma et al., 2012, Qu et al., 2008, Yan et al., 2009). However, it has also been reported that the levels of fluoride used in preventing dentin hypersensitivity might initiate apoptosis (Jacinto-Alemán et al., 2010). A even lower concentration of fluoride can selectively affect odontoblast gene expression ex vivo (Wurtz et al., 2008) and down-regulate dentine sialophosphoprotein (DSPP) mRNA in vivo. Fluoride-induced abnormalities in the dentine of deer teeth is characterized by the presence of interglobular dentine and regular bands of hypo- and hypermineralized dentine and giant tubules (Richter et al., 2010). Thus, the pro-apoptotic effect of fluoride might be key contributor to dentine abnormalities induced by high concentrations of NaF (Lyaruu et al., 2008). The purpose of this study was to evaluate the cytotoxic and apoptotic effects of NaF on odontoblast cells and to clarify the underlying mechanism of these effects.

Section snippets

Chemicals

Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM) and trypsin were obtained from Gibco (Grand Island, NY, USA). Methyl thiazolyl tetrazolium (MTT), sodium fluoride (NaF), dimethyl sulfoxide (DMSO) and Hoechst 33258 were obtained from Sigma–Aldrich Co. (St. Louis, MO, USA).

Cell culture

Odontoblast-lineage cells (OLC), a novel odontoblast-lineage cell line established from mouse embryo tooth germs in E18.5 mouse mandibles, kindly provided by Dr. Arany, (Arany et al., 2006). OLCs were

NaF decreases cell viability and induces apoptosis in odontoblast cells

The cytotoxicity of NaF was determined by the MTT assay. As shown in Fig. 1a, NaF is cytotoxic to OLC cells in a dose-and time-dependent manner when administered at concentrations ranging from 1 to 6 mM for 6 to 48 h (Fig. 1a). Reduction of cell viability reached approximately 50% after 24 h treatment with 4 mM NaF.

Apoptosis of OLC cells induced by NaF was assessed by in situ TUNEL assay. A large number of TUNEL-positive cells were present in cells exposed to 4 mM NaF (Fig. 1b), and quantitative

Discussion

In this study, we demonstrate that fluoride induces apoptosis in odontoblasts through a JNK/ERK-mediated mitochondria-dependent pathway. In accordance with our previous study, fluoride is cytotoxic to OLC cells in a concentration- and time-dependent manner. Meanwhile, characteristics of apoptosis, including TUNEL-positive staining, morphological and ultrastructural changes and expression of cleaved caspase-3 are observed in OLC cells exposed to 4 mM NaF, demonstrating that fluoride induces

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by the China National Natural Science Foundation (31271048, 81170948 and 31200738).

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    These authors contributed equally to the paper.

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