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In this study, experimental pathology, flow cytometry (FCM), quantitative real-time polymerase chain reaction (qRT-PCR), and western blot (WB) were used to evaluate the effects of sodium fluoride (NaF) on hepatocellular cell cycle progression in mice. A total of 240 ICR mice were divided equally into four groups; the experimental groups received 12, 24, or 48 mg/kg NaF intragastrically for 42 days, while the control group received distilled water. Doses of NaF above 12 mg/kg increased the percentage of cells in S phase (S-phase arrest), reduced percentages of cells in G0/G1 or G2/M phase, and activated the ATM-p53-p21 and ATR-Chk1-Cdc25A pathways. Activation of these pathways was characterized by up-regulation of ATM, p53, p21, ATR, and Chk1 mRNA and protein expression, and down-regulation of Cdc25A, cyclin E, cyclin A, CDK2, CDK4, and proliferating cell nuclear antigen (PCNA) mRNA and protein expression. These results indicate that NaF caused S-phase arrest by activating the ATM-p53-p21 and ATR-Chk1-Cdc25A pathways.

// Hongrui Guo 1, * , Ping Kuang 1, * , Qin Luo 1, * , Hengmin Cui 1, 2 , Huidan Deng 1 , Huan Liu 1 , Yujiao Lu 1 , Jing Fang 1, 2 , Zhicai Zuo 1, 2 , Junliang Deng 1, 2 , Yinglun Li 1, 2 , Xun Wang 1, 2 and Ling Zhao 1, 2 1 College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, China 2 Key Laboratory of Animal Diseases and Environmental Hazards of Sichuan Province, Sichuan Agriculture University, Ya’an 625014, China * These authors have contributed equally to this work Correspondence to: Hengmin Cui, email: cui580420@sicau.edu.cn Keywords: NaF, blood, cellular immunity, humoral immunity, mice Received: March 21, 2017 Accepted: July 19, 2017 Published: August 10, 2017 ABSTRACT Exposure to high fluorine can cause toxicity in human and animals. Currently, there are no systematic studies on effects of high fluorine on blood cellular immunity and humoral immunity in mice. We evaluated the alterations of blood cellular immunity and humoral immunity in mice by using flow cytometry and ELISA. In the cellular immunity, we found that sodium fluoride (NaF) in excess of 12 mg/Kg resulted in a significant decrease in the percentages of CD3 + , CD3 + CD4 + , CD3 + CD8 + T lymphocytes in the peripheral blood. Meanwhile, serum T helper type 1 (Th1) cytokines including interleukin (IL)-2, interferon (IFN)-γ, tumor necrosis factor (TNF), and Th2 cytokines including IL-4, IL-6, IL-10, and Th17 cytokine (IL-17A) contents were decreased. In the humoral immunity, NaF reduced the peripheral blood percentages of CD19 + B lymphocytes and serum immunoglobulin A (IgA), immunoglobulin G (IgG) and immunoglobulin M (IgM). The above results show that NaF can reduce blood cellular and humoral immune function in mice, providing an excellent animal model for clinical studies on immunotoxicity-related fluorosis.

The current study was conducted to investigate the effect of sodium fluoride (NaF) on the oxidative stress and apoptosis as well as their relationship in the mouse liver by using methods of flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR), western blot, biochemistry and experimental pathology. 240 four-week-old ICR mice were randomly divided into 4 groups and exposed to different concentration of NaF (0 mg/kg, 12 mg/kg, 24 mg/kg and 48 mg/kg) for a period of 42 days. The results showed that NaF caused oxidative stress and apoptosis. NaF-caused oxidative stress was accompanied by increasing reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and decreasing mRNA expression levels and activities of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (GSH-PX) and glutathione-s-transferase (GST). NaF induced apoptosis via tumor necrosis factor recpter-1 (TNF-R1) signaling pathway, which was characterized by significantly increasing mRNA and protein expression levels of TNF-R1, Fas associated death domain (FADD), TNFR-associated death domain (TRADD), cysteine aspartate specific protease-8 (caspase-8) and cysteine aspartate specific protease-3 (caspase-3) in dose- and time-dependent manner. Oxidative stress is involved in the process of apoptotic occurrence, and can be triggered by promoting ROS production and reducing antioxidant function. NaF-caused oxidative stress and apoptosis finally impaired hepatic function, which was strongly supported by the histopathological lesions and increased serum alanine amino transferase (ALT), aspartic acid transferase (AST), alkaline phosphatase (AKP) activities and TBIL contents.

// Ping Kuang 1, * , Huidan Deng 1, * , Hengmin Cui 1, 2 , Lian Chen 1 , Jing Fang 1, 2 , Zhicai Zuo 1, 2 , Junliang Deng 1, 2 , Xun Wang 1, 2 , Ling Zhao 1, 2 1 College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, China 2 Key Laboratory of Animal Diseases and Environmental Hazards of Sichuan Province, Sichuan Agricultural University, Ya’an 625014, China * These authors contributed equally to this work Correspondence to: Hengmin Cui, email: cui580420@sicau.edu.cn Keywords: NaF, growth index, T and B-cell, cytokine, cell cycle Received: November 07, 2016 Accepted: December 07, 2016 Published: December 16, 2016 ABSTRACT At present, very limited studies focus on the toxic effect of sodium fluoride (NaF) on splenic development of human and animals in vivo . This study was firstly designed to evaluate the toxic effects of NaF on the splenic development of mice in vivo by observing histopathological lesions, changes of splenic growth index (GI), T and B cells, immunoglobulin A (IgA), immunoglobulin G (IgG) and immunoglobulin M (IgM) contents, cytokine protein expression levels, and cell cycle and cyclins/cdks protein expression levels using the methods of pathology, flow cytometry (FCM), western blot (WB), and enzyme-linked immunosorbent assay (ELISA). A total of 240 ICR mice were equally allocated into four groups with intragastric administration of distilled water in the control group and 12, 24, 48 mg/kg NaF solution in the experimental groups for 42 days. The results showed that NaF in 12 mg/kg and over caused the toxic effects on splenic development, which was characterized by reducing growth index and lymphocytes in the white and red pulp histopathologically, increasing cell percentages of the G0/G1 phase and decreasing cell percentages of the S phase, and reducing T cells and B cells as well as IgA, IgG, and IgM contents when compared with those in the control group. Concurrently, cytokines including interleukin-2 (IL-2), transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ) and cyclin (E/D and CDK2/4) protein expression levels were markedly decreased ( P < 0.05 or P < 0.01), and interleukin-10 (IL-10) protein expression levels were significantly increased ( P < 0.05 and P < 0.01) in the three NaF-treated groups. Toxic effects finally impaired the splenic cellular immunity and humoral immunity due to the reduction of T and B cell population and activity. Cell cycle arrest is the molecular basis of NaF-caused toxic effects on the splenic development.

// Ping Kuang 1, * , Huidan Deng 1, * , Hengmin Cui 1, 2 , Lian Chen 1 , Hongrui Guo 1 , Jing Fang 1, 2 , Zhicai Zuo 1, 2 , Junliang Deng 1, 2 , Xun Wang 1, 2 , Ling Zhao 1, 2 1 College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, China 2 Key Laboratory of Animal Diseases and Environmental Hazards of Sichuan Province, Ya’an 625014, China * These authors have contributed equally to this work Correspondence to: Hengmin Cui, email: cui580420@sicau.edu.cn Keywords: sodium fluoride, cytokine, cell cycle, splenic T lymphocytes, splenic B lymphocytes Received: May 01, 2016 Accepted: July 28, 2016 Published: August 16, 2016 ABSTRACT Fluoride-induced immunotoxicity has been documented in vivo, but limited reports have focused on the effects of fluoride on lymphocytes in vitro. Therefore, we have examined the suppressive effects of sodium fluoride on cultured splenic lymphocytes in mice. CD3+ T lymphocytes, CD19+ B lymphocytes, cytokines, and cell-cycle markers were analyzed through the use of a cell-counting kit, western blot, and flow cytometery. Splenic lymphocytes were isolated from 3-week-old male ICR mice and exposed to sodium fluoride (0, 100, 500, and 1000 μmol/L) for 24 h. The percentages of CD3 + , CD3 + CD4 + , CD3 + CD8 + T lymphocytes and CD19 + B lymphocytes were decreased (P