Fluoride induces apoptosis and alters collagen I expression in rat osteoblasts
Introduction
Fluoride (F) is considered one of the most essential elements for maintaining the normal cellular processes in an organism. However, prolonged and excessive F intake can contribute to a serious public health problem known as fluorosis, characterized by tooth discoloration and skeletal manifestations such as crippling, osteoporosis, and osteosclerosis. Endemic fluorosis is now known to be a global issue, and understanding the pathogenesis of this disease marks for scientific investigation (Bailey et al., 2006). At low concentrations, F plays an important role in order to promote growth, development, and maintenance for the skeletal system. However, at high concentrations, F can cause damage to multiple organs and tissues especially to the skeleton and teeth, progressing to fluorosis (Krishnamachari, 1986). In the process of skeletal fluorosis, F affects all types of cells that participate in bone turnover, notably the osteoblasts (OB) and osteoclasts. Fluorosis not only causes damage to DNA, but also decreases DNA replication, as shown by changes in the cell cycle (Yu et al., 2001, Zhong et al., 2005). Cell cycle regulation is one of the key regulatory mechanisms of cell growth (Gamet-Payrastre et al., 2000). Many cytotoxic and genotoxic agents can arrest the cell cycle at different phases and then induce apoptotic cell death (Orren et al., 1997, Fujimoto et al., 1999). Many investigations have previously demonstrated that F is a cytotoxic agent inducing apoptosis, and disrupting cell cycle progression in many types of cells such as neurons (Ge et al., 2006), primary rat hippocampal neurons (Zhang et al., 2008), testicular cells (Huang et al., 2007a, Huang et al., 2007b), and OB (Wang et al., 2001).
Moreover, OB are the primary cells contributing to bone formation. OB actively secrete type I collagen, growth factors, enzymes, and minerals into the bone matrix (Goltzman, 2002). Type I collagen, the main collagen type expressed in bone (Nicolai et al., 2004), consists of two α1(I) and one α2(I) polypeptide chains that assemble into the functional collagen protein. The α1(I) and α2(I) subunits are encoded as COL1A1 and COL1A2, respectively, and have different primary amino acid sequences (Miu et al., 2002, Xu et al., 2003). It is well known that collagen is a target of excessive F exposure (Susheela and Sharma, 1982, Pu et al., 1996). Many studies have shown that F can negatively affect collagen metabolism in cartilage and bone (Pu et al., 1996, Guo et al., 2002). Our previous experiments have demonstrated significant negative effects of F on type I collagen (COL1A1) in the ribs of rabbits, and the teeth of sheep and guinea pigs (Li et al., 2007, Yan et al., 2007, Han et al., 2010, Wang et al., 2010). However, whether F has negative effects on the expression of both COL1A1 and COL1A2 genes in vitro is unclear. It is of interest to determine whether the decrease in collagen I is a result of protein degradation or due to an inhibition of protein synthesis, happening either at the level of transcription or translation. Previous evidence indicates that sodium fluoride (NaF) interferes with the maturation and normal metabolism of tissue collagen due to defective fibers that are produced during fluoride toxicity providing inadequate cross-links (Sharma, 1982). Thus, investigating the effect of F on collagen protein by quantifying the expression of COL1A1 and COL1A2 will allow for a better understanding behind the molecular mechanisms of F toxicity.
In this study, we investigate the effect of F on OB survival and expression levels of COL1A1 and COL1A2 of OB, providing basic data for further elucidating the molecular mechanisms of skeletal damage induced by F toxicity.
Section snippets
Materials and chemicals
Ten 1-day-old female neonatal Wistar rats were provided by the Experimental Animal Center of Shanxi Medical University of China. Dulbecco's Modified Eagle Medium (DMEM) and trypsin were supplied by Gibco Company (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Hangzhou Sijiqing Biological Engineering Material Company (Hangzhou, China). 2-[4-(2-Hydroxyerhyl)-1-piperazinyl] ethanesulfonic acid (HEPES) buffer was obtained from Hyclone Company of America. NaF and MTT were
Effect of NaF on OB morphology
After treatment with various concentrations NaF for 72 h, we used an inverted microscope to evaluate the cellular morphology of the OB. As shown in Fig. 1, while a good growth state of the control group was observed, the cytoplasm and nucleus of some of the OB were affected due to the certain concentrations of F administered. The majority of OB became curled, flaked, contracted, and suspended in the media with a dosage of 20 mg/L of NaF.
Effect of NaF on OB cell cycle
OB were incubated for 72 h under different dosages of NaF in
Discussion
In the past, F was considered to be an essential element. In actuality, there is a lack of agreement as to whether the role of F in human nutrition is optimal for development and growth (Nielsen, 2009). Additional risks of increased F exposure are the effects on bone cells (both OB and osteoclasts) that can lead to the development of skeletal fluorosis (NRC, 2006). A majority of studies indicate that a low concentration of F stimulates OB proliferation and differentiation, while a high
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
This research was sponsored by the China National Natural Science Foundation (Grant No. 30871899) and the Shanxi Province Key Laboratory Open Foundation (Grant No. 20081057).
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