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Previous studies have reported that excessive fluoride exposure induced liver damage. However, the underlying mechanism of fluoride-induced hepatic toxicity is still unclear. Hence, this study was aimed to evaluate the fluoride-induced apoptosis, autophagy, and IL-17 signaling pathway-related genes to explore the possible mechanisms of NaF-induced liver injury in mice. For this, 48 male mice were allotted randomly to four groups, treated with deionized water, 25, 50, 100 mg/L NaF for 150 days continuously. Our results suggested that treatment with NaF decreased the PAS staining-positive area, with a concomitant increase in liver score, and serum ALT and AST levels which indicated that NaF induced the liver injury. In addition, the qRT-PCR, immunohistochemistry, and western blotting results indicated that NaF exposure activated IL-17 signaling, apoptosis, and autophagy pathways. In summary, these results suggested that NaF induced apoptosis and autophagy in liver by activating the IL-17 signaling pathway, eventually leading to impaired liver function.

For the past decade, there has been an increased concern about the health risks from arsenic (As) exposure, because of its neurotoxic effects on the developing brain. The exact mechanism underlying As-induced neurotoxicity during sensitive periods of brain development remains unclear, especially the role of blood-brain barrier's (BBB) tight junction (TJ) proteins during As-induced neurotoxicity. Here, we highlight the involvement of TJ proteins in As-induced autophagy in cerebral cortex and hippocampus during developmental periods [postnatal day (PND) 21, 28, 35 and 42]. Here, the administration of arsenic trioxide (As 2 O 3 ) at doses of 0.15 mg or 1.5 mg or 15 mg As 2 O 3 /L in drinking water from gestational to lactational and continued to the pups till PND42 resulted in a significant decrease in the mRNA expression levels of TJ proteins (Occludin, Claudin, ZO-1 and ZO-2) and Occludin protein expression level. In addition, As exposure significantly decreased PI3K, Akt, mTOR, and p62 with a concomitant increase in Beclin1, LC3I, LC3II, Atg5 and Atg12. Moreover, As exposure also significantly downregulated the protein expression levels of mTOR with a concomitant upregulation of Beclin 1, LC3 and Atg12 in all the developmental age points. However, no significant alterations were observed in low and medium dose-exposed groups of PND42. Histopathological analysis in As-exposed mice revealed decreased number of pyramidal neurons in hippocampus; and neurons with degenerating axons, shrinkage of cells, remarkable vacuolar degeneration in cytoplasm, karyolysis and pyknosis in cerebral cortex. Ultrastructural analysis by transmission electron microscopy revealed the occurrence of autophagosomes and vacuolated axons in the cerebral cortex and hippocampus of the mice exposed to high dose As at PND21 and 42. The severities of changes were found to more persist in the cerebral cortex than in the hippocampus of As-exposed mice. Finally, we conclude that the leaky BBB in cerebral cortex and hippocampus may facilitate the transfer of As and induces autophagy by inhibiting PI3K/Akt/mTOR signaling pathway in an age-dependent manner, i.e., among the four different developmental age points, PND21 animals were found to be more vulnerable to the As-induced neurotoxicity than the other three age points.

It is well known that high fluoride results in low fertility. Epididymis is the important place for spermatozoa maturation, which is essential for successful fertilization. In the previous studies, fluoride was reported to damage the epididymal structure of mouse and rabbit. However, the mechanism underlying sodium fluoride (NaF)-induced epididymal toxicity has not yet been well elucidated. The aim of this study is to explore the global protein alterations in epididymis of mice exposed to NaF using the iTRAQ technique. Results showed that 211 proteins were differentially expressed in both 25 and 100 mg/L NaF groups. Some of them have been proved to be important for reproduction, such as low-density lipoprotein receptor-related protein 2 (Lrp2), cytochrome c, testis-specific (Cyct), sorbitol dehydrogenase (Sord), glutathione S-transferases (GSTs), acrosin, beta-defensin 126, cysteine-rich secretory protein (Crisp) 1, and Crisp2. Gene ontology (GO) analysis suggested cellular process, organelle and catalytic activity account for high percent and number of differentially expressed proteins. 171 pathways were found after the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, among which the representative maps, such as ribosome, focal adhesion, and phagosome, were involved. Different functional categories post-translational modification, protein turnover, chaperones; translation, ribosomal structure and biogenesis; cytoskeleton; energy production and conversion are implicated in the Cluster of Orthologous Groups (COG) of proteins analysis. Subsequently, the effect of NaF on the antioxidant activity in epididymis, especially glutathione and glutathione-related enzymes, was evaluated. Results exhibited high fluoride caused low total antioxidant capacity (T-AOC), high methane dicarboxylic aldehyde (MDA), decreased reduced glutathione (GSH), and the glutathione-related enzymes [GSH peroxidase (GPx), GSH reductase (GR), and GSH S-transferase (GST)] changes in activity, protein, and mRNA expressions. In summary, NaF decreased the antioxidant activity of epididymis, especially glutathione and glutathione-related enzymes, as well as iTRAQ results, providing new explanations for the low sperm quality induced by fluoride.

Titanium dioxide nanoparticles (nano-TiO2) are frequently used in cosmetics, paints, sunscreens and the like. Recent studies have demonstrated that nano-TiO2 might be deleterious for the male reproductive function. However, the effects of pubertal nano-TiO2 exposure on testosterone (T) synthesis and spermatogenesis remained to be elucidated. Here, we investigated the effect of pubertal nano-TiO2 exposure on the synthesis of T and spermatogenesis. Nano-TiO2 was orally administered daily to Kunming male mice from 28th postnatal day (PND 28) to PND 70. The percentage of spermatozoa abnormality in epididymides was markedly increased in mice exposed to nano-TiO2; decreased layers of spermatogenic cells and vacuoles in seminiferous tubules were also observed in the nano-TiO2 treated group. In addition, pubertal nano-TiO2 exposure significantly decreased the serum T levels in male mice. Moreover, mice exposures to nano-TiO2 significantly reduced the expression of 17β-hydroxysteroid dehydrogenase and P450 17α-hydroxysteroid dehydrogenase in the testis of mice, while the expression of cytochrome P450-19, a key enzyme for the translation of T to estradiol (E2), was increased. Taken together, these results indicated that nano-TiO2 could influence the levels of serum T through changes in both the synthesis and translation of T. Furthermore, the decreased serum T synthesis might contribute to the reduced spermatogenesis in mice exposed to nano-TiO2.

Reproductive process covers lots of procedures, including capacitation, hyperactivation, chemotaxis and the acrosome reaction. Each plays an important role in the success of fertilization. Although multiple studies have reported the toxic effects of fluoride on the male reproduction, the effect of fluoride on sperm chemotaxis is little known. This study is to examine the effect of fluoride on the sperm chemotaxis and then to reveal the underling mechanisms of fluoride toxicity in sperm chemotaxis. 260 healthy Kunming male mice (8 weeks old) were randomly divided into four groups and exposed to 50, 100, 150 mg NaF/L in the drinking water for 8 weeks. At the end of the exposure, sperm chemotaxis was examined using a microchannel-based device. Ca(2+) concentration, adenylate cyclase (AC) content and mRNA expression of mACIII, mACVIII, Golf alpha, CatSper1, CatSper2 were measured to elucidate the possible molecular mechanisms. The results showed that the percentage of chemotactic sperm was decreased by NaF in a dose-dependent manner. In the 100 and 150 mg/L groups, Ca(2+) concentration and AC content were notably lower than the control group. Compared with the control group, mRNA expression of CatSper1 in the 100 and 150 mg/L treatment groups was decreased significantly, and other genes showed no statistical difference. These data suggested that excessive fluoride did adversely affect sperm chemotaxis. The alteration of Ca(2+) concentration, AC content and CatSper1 mRNA expression level may play a key role in the mechanism underlying the affection.

Epidemiological and experimental studies have demonstrated the atherogenic effects of environmental toxicant arsenic and fluoride. Inflammatory mechanism plays an important role in the pathogenesis of atherosclerosis. The aim of the present study is to determine the effect of chronic exposure to arsenic and fluoride alone or combined on inflammatory response in rabbit aorta. We analyzed the expression of genes involved in leukocyte adhesion [P-selectin (P-sel) and vascular cell adhesion molecule-1(VCAM-1)], recruitment and transendothelial migration of leukocyte [interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1)] and those involved in pro-inflammatory cytokines [interleukin-6 (IL-6)]. We found that fluoride and arsenic alone or combined increased the expression of VCAM-1, P-sel, MCP-1, IL-8, and IL-6 at the RNA and protein levels. The gene expressions of inflammatory-related molecules were attenuated when co-exposure to the two toxicants compared with just one of them. We also examined the lipid profile of rabbits exposed to fluoride and (or) arsenic. The results showed that fluoride slightly increased the serum lipids but arsenic decreased serum triglyceride. We showed that inflammatory responses but not lipid metabolic disorder may play a crucial role in the mechanism of the cardiovascular toxicity of arsenic and fluoride.

Exposure to fluoride can induce low sperm quality; however, little is known about the molecular mechanisms by which fluoride exerts its toxic effects. This study was conducted to evaluate ultrastructure, oxidative stress, and apoptosis in sperm of mice treated with 150 mg/l NaF for 49 days. Furthermore, microarray analysis was also utilized to characterize the effects of fluoride in gene expression profiling on mice sperm. An increased ROS and a decreased TAC accompanied with distinct morphological changes and significant apoptosis were observed in mice sperm from the fluoride group. Fluoride exposure also significantly elevated the protein expressions of cytochrome c and active caspase-3. In global gene expression profiling, 34 up-regulated and 63 down-regulated genes, which are involved in several sperm biological processes including signal transduction, oxidative stress, apoptosis, electron transport, glycolysis, chemotaxis, spermatogenesis, and sperm capacitation, were significantly differentially expressed. Based on these findings, it was proposed that oxidative stress induced by excessive ROS may trigger sperm apoptosis through mitochondrial impairment, resulting in decreased fertility in mice exposed to fluoride. Microarray analysis also provided several important biological clues for further investigating fluoride-induced damage in sperm morphology and functions.

Epidemiological investigations reveal that high fluoride and low iodine have strong adverse effects on the intelligence quotient (IQ) of children. Studies also report that in some high fluoride areas, iodine deficiency also exists, especially in China. Here, with the proteomic techniques, we first report on the proteomic changes in brain proteins in offspring rats at postnatal day 20 exposed to high fluoride and/or low iodine. To investigate molecular mechanisms of central neural system injury induced by the above two elements, proteins were isolated and profiled by two-dimensional gel electrophoresis (2DE). By the analysis of Image-Master 2D Elite software, 71 protein spots in 2DE gels of treatment groups were gained and up- or down-regulated by two folds, and 5 proteins were regulated by five folds, with the comparison to the control group. The proteins changed by five folds were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The identified proteins are mainly related with cellular signaling, energy metabolism, and protein metabolism and provide a valuable clue to explore the mechanism underlining the neurotoxicity of high fluoride and low iodine. Moreover, these results could provide potential biomarkers for hazards caused by excessive fluoride and low iodine.

Sperm hyperactivation is crucial for a successful fertilization; however, the influence of fluoride (F) to hyperactivation is still in its infancy. The purpose of this study was to investigate the effect of sodium fluoride (NaF) on sperm hyperactivation, Ca2+/CALM-CAMK2 signaling, and CatSper1 and CatSper2 mRNA expression in mice sperm. Adult male Kunming mice were administrated with 30, 70, and 150 mg NaF/l (corresponding to 2.84 +/- 0.29, 6.28 +/- 0.61, and 14.18 +/- 1.00 mg F/kg body weight per day) through drinking water for 49 days. The results showed that NaF reduced the sperm hyperactivated motility in a dose-dependent manner. Compared with the controls, intracellular Ca2+ concentration and CAMK2 protein were significantly decreased in mice treated with 70 and 150 mg NaF/l, while no effect on CALM was determined in all treatment groups. Furthermore, decreased sperm CatSper1 mRNA expression was also observed in response to middle and higher doses of NaF (70, 150 mg/l) with comparison to the control group, whereas no change in the mRNA expression of CatSper2 was detected in NaF administrated groups. Treatment with 30 mg NaF/l exhibited slight effects on the above indexes with no statistical difference. These findings indicated that exposure to 70 and 150 mg/l NaF for 49 days could result in low hyperactivation via alteration of Ca2+ signaling pathway involving CatSper1 in sperm from mice.

Long-term excessive fluoride intake is linked to skeletal disease. Skeletal health is influenced by the balance between bone formation and resorption of which osteoblast function is critical. The objectives of this study were to determine the effect of fluoride treatment on osteoblast proliferation, apoptosis and caspase-3 and caspase-9 mRNA expression in vitro. Neonatal rat osteoblasts were cultured in the presence of varying concentrations (0.5-30 mg/l) of sodium fluoride and effects of treatments were determined. Treatment with sodium fluoride inhibited osteoblast proliferation in a dose-dependent fashion and effects were maximal after 120 h incubation. A significant increase in osteoblast apoptosis was observed (after 24 and 72-h treatment) in response to the lowest dose of sodium fluoride (0.5 mg/l) and osteoblast apoptosis was further increased in response to higher doses. Increased-osteoblast caspase-3 and caspase-9 mRNA was also observed in response to sodium fluoride treatment (5 mg/l) for 72 h. Results indicate that negative effects of excess fluoride on skeletal health may be mediated in part by inhibition of osteoblast survival.