Effects on protein and mRNA expression levels of p53 induced by fluoride in human embryonic hepatocytes
Introduction
Fluorosis, which seriously impairs human health, is prevalent in some parts of central and western China. Epidemiological evidence and experimental results have shown that fluorosis does not only cause adverse biochemical effects on structure and function of skeletal and teeth, but also of non-skeletal systems such as brain, liver, renal, and spinal cord. More recent studies have suggested that the toxicity of excessive fluoride is relative to apoptosis and can induce a variety of apoptosis cell types (Anuradha et al., 2001, Chen et al., 2002, Refsnes et al., 2003, Tokunaga et al., 2003, Wang et al., 2004). Although apoptosis induced by fluoride has become an important mechanism of fluoride toxicity (Zhang and Wu, 2003), the real apoptosis mechanism induced by fluoride is still unknown. Furthermore, there are conflicting reports with regard to excessive fluoride causing cancer. Some animal experimental results indicated that excessive fluoride could cause cancer (Toft, 1989), which has also been supported by epidemiological investigations (Persing, 1989).
The p53 protein, a tumor suppressor, possesses a potent transactivation domain which regulates the transcription of a number of genes. The activities of some of these genes are associated to cell proliferation, cell differentiation, and DNA repair systems. The p53 protein acts as a “molecular police” and a critical molecular of these functions (Hall et al., 1996, Lane, 1992). When cells are subjected to DNA damage, the p53 protein binds with DNA, RNA polymerase and then regulates the expression of related genes as a nuclear transcriptional factor in order to regulate the cell cycle and promote cell apoptosis to maintain the integrity of the cellular genome. Some animal experiments have shown that p53 regulates cell apoptosis induced by fluorosis (Jiang, 1999, Hu et al., 2002). Moreover, wild type p53 acts as a tumor suppressor protein, whereas mutant p53 could increase function properties such as immortalization of primary tumor cells. Change or absence of wild type p53 was associated with comprehensive human tumors (Hollstein et al., 1991, Levine et al., 1991). The p53 gene mutation has been found in approximately 40%–50% of different human cancer cells (Vogelstein and Kinzler, 1992). Ramesh et al. (2001) reported that p53 mutation was found in patients with osteosarcoma with a high bone fluoride level (Ramesh et al., 2001). Due to the various effects of p53 in cell apoptosis and p53 mutation in tumor cells, we investigated the effects of different doses of sodium fluoride on protein and mRNA expression levels in human L-02 cells in vitro.
Section snippets
General chemicals
We obtained L-02 cells from China Type culture collection at the Wuhan University and Fetal bovine serum, HEPES buffer, and DMEM cell culture powder were obtained from GIBCO BRL (Paisley, Scoltland). Trypsin, DAB, biascrylamide, RNAase, and HRP-conjugated sheep anti-mouse antibodies were purchased from Sigma (Diesenhofen, Germany), and acrylamide as well as protein marker from Life Technologies GmbH (Karlsruhe, Germany). RevertAid™ First Strand cDNA Synthesis Kit, dNTP Mix, Taq enzyme, and p53
Effect of fluoride on L-02 cell viability
Table 1 demonstrates that the survival rate of L-02 treated cells by high dose of fluoride was significantly lower than in the control group (P < 0.01), although there was no statistical difference of cell viability between the low dose and middle dose compared with the control group (P > 0.05).
Effects of fluoride on p53 protein expression level in L-02 cells
Fig. 1 shows the protein expression level of p53 in L-02 cells, which is exposed to different doses of sodium fluoride. Results from densitometric analyses of the intensity of the various bands are shown in
Discussion
More recent studies have found that p53 can mediate various gene expressions such as p21, c-myc, bcl-2, TGF-β, IL-2, fas, and bax, and that it also plays an important role in cell proliferation and differentiation (Jiang, 1999). Furthermore, p53 acts as a key-regulating molecule during cell stress, which could cause different responses such as suppressing cell growth or apoptosis to cell emergency signals by transcription or non-transcription (Hall et al., 1996). When cells were subjected to
Acknowledgements
The authors would like to thank Ms. S. Albrecht very much for the edition of the manuscript into proper English. The work was supported by grants from the National Nature Science Foundation of China (Nos. 30271155, and 30371250), and the China national key basic research and development program (No. 2002CB512908).
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2016, Fish and Shellfish ImmunologyCitation Excerpt :Fluoride is also well-known as the element producing a variety of cellular and metabolic changes e.g. glycolysis inhibition, changes in membrane receptors and apoptotic induction. Supplement of NaF as the main source of fluoride to mammalian cells can induce apoptosis both in vitro and in vivo via the reactive oxygen species (ROS)-dependent, caspase-mediated and JNK signalling pathways [4,22–24]. Morphological changes and analysis of Annexin V, a well-known early apoptotic detection marker [25], and TUNEL, the outstanding detection assay of late apoptosis [9,26], including the investigation of the expression level of IAP and caspase-3 genes by real-time PCR technique, were undertaken to indicate the cellular death pattern of primary haemocytes in this study.
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Present address: Fresenius Biotech GmbH, Division of Cell Therapy, Bad Homburg 61352, Germany.