Background/Aims: Excessive fluoride intake can induce cytotoxicity, DNA damage and cellcycle changes in many tissues and organs, including the kidney. However, the underlying molecular mechanisms of fluoride-induced renal cell-cycle changes are not well understood at present. In this study, we used a mouse model to investigate how sodium fluoride (NaF) induces cell-cycle changes in renal cells. Methods: Two hundred forty ICR mice were randomly assigned to four equal groups for intragastric

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... istration of NaF (0, 12, 24 and 48 mg/kg body weight/day) for 42 days. Kidneys were taken to measure changes of the cell-cycle at 21 and 42 days of the experiment, using flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot methods. Results: NaF, at more than 12 mg/kg body weight, induced G2/M phase cell-cycle arrest in the renal cells, which was supported by the finding of significantly increased percentages of renal cells in the G2/M phase. We found also that G2/M phase cell-cycle arrest was accompanied by up-regulation of p-ATM, p-Chk2, p-p53, p-Cdc25C, p-CDK1, p21, and Gadd45a protein expression levels; up-regulation of ATM, Chk2, p53, p21, and Gadd45a mRNA expression levels; down-regulation of CyclinB1, mdm2, PCNA protein expression levels; and down-regulation of CyclinB1, CDK1, Cdc25C, mdm2, and PCNA mRNA expression levels. Conclusion: In this mouse model, NaF, at more than 12 mg/ kg, induced G2/M phase cell-cycle arrest by activating the ATM-Chk2-p53/Cdc25C signaling Mouse pathway, which inhibits the proliferation of renal cells and development of the kidney. Activation of the ATM-Chk2-p53/Cdc25C signaling pathway is the mechanism of NaF-induced renal G2/M phase cell-cycle arrest in this model. Introduction Fluoride is a ubiquitous ingredient of drinking water, foodstuffs, medicines and dental products [1, 2] , and it is widely used in a variety of industrial practices, such as the production of phosphoric acid, phosphate fertilizers, ceramics, aluminum, steel, and brick [3] . Moreover, NaF is a useful phosphatase inhibitor. According to World Health Organization guidelines, the recommended concentration of fluoride in drinking water is 1.0 mg/L, and the upper limit is 1.5 mg/L [4]. Especially high fluoride concentrations in nature have been found in India, Mexico, Turkey, North Africa and China (Yunnan, Gui zhou) [5] . Excessive fluoride intake can cause dental and skeletal fluorosis in animals and humans [6, 7] as well as structural and functional changes in soft tissues, including brain [8-10], thymus [11, 12], liver [13-15], heart [11, 16], lung [17], spleen [18-21], intestine [22, 23], cecal tonsil [24, 25], reproductive organs [26] and the bursa of Fabricius [27] . As the major organ involved in retention and excretion of fluoride, the kidney is sensitive to fluoride toxicity [28] . And previous studies have demonstrated that high doses of fluoride can induce cytotoxicity, lipid peroxidation, DNA damage, apoptosis and cell-cycle changes in the kidney [29, 30] . The cell-cycle is a complex process that participates in the growth and proliferation of cells, development of organisms, regulation of DNA damage repair, and diseases such as cancer [31] . The divisions of the standard cell-cycle are G1 (cell prepares for DNA synthesis), S (cell is synthesizing DNA), G2 (cell prepares for mitosis), and mitosis (M) phases [32] . Cells in the G1 phase can enter a resting state called the G0 phase, which usually indicates potential for cell division. Cell transition from one cell-cycle phase to another occurs in an orderly fashion and is regulated by cell-cycle-regulating proteins, such as the cyclin proteins and cyclin-dependent kinases (CDKs) [33] . Checkpoints also exist in the cell-cycle to ensure correct cell-cycle progression. Cell-cycle checkpoints are mainly positioned before the cell enters S phase (G1/S checkpoint), after DNA replication (G2/M checkpoint) or during S phase (S checkpoint) [34] . In response to DNA damage, cell-cycle checkpoints arrest the cell-cycle progression to provide more time for DNA repair [35] . The prolonged cell-cycle arrest disrupts the balance between cell proliferation and cell death, and ultimately results in cellular growth arrest or death [36] . Bai et al. [37] have reported that excessive sodium fluoride (NaF) intake can induce G0/G1 phase cell-cycle arrest of renal cells in chickens. Also, NaF, at the dose of 50 mg/L, reduced the number of rat renal cells in G2/M phase [38] , and excessive NaF induced loss of control in proliferation of rat renal cells by disturbing the G1/S and G2/M phase [39] . However, there are inconsistencies in these reports about the effects of fluoride on the renal cell-cycle. The cell-cycle is controlled by numerous mechanisms that ensure correct cell division. Ataxia-telangiectasia-mutated (ATM), as a sensor of DNA damage, plays an important role in cell proliferation and DNA repair [40] . Recent studies have reported that ATM-dependent signaling pathways participate in the regulation of multiple cell-cycle checkpoints, and the activation of ATM-dependent signaling pathways can induce the expression of many genes that are involved in cellular functions, such as cell-cycle arrest, DNA repair, or apoptosis [40] . However, there are no studies on the molecular mechanisms of fluoride-induced renal cellcycle changes in animals and humans. In this study, we first used a mouse model to investigate, by flow cytometry, the effects of fluoride on the cell-cycle in renal cells. We then explored the underlying molecular mechanisms of NaF-induced renal cell-cycle changes by measuring protein expression levels of phosphor-ATM (p-ATM, Ser-1981), p-checkpoint kinase 2 (p-Chk2, Thr-68), p-p53 (Ser-15), p21, growth arrest and DNA-damage-inducible protein 45 alpha (Gadd45a), Cyclin B 1 , p-CDK1 (Tyr-15), p-cell division cyclin 25C (p-Cdc25C, Ser-216), murine double minute 2