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Background: Although the changes of mitogen-activated protein kinase (MAPK) pathway in the central nervous system (CNS) induced by excessive fluoride has been confirmed by our previous findings, the underlying mechanism(s) of the action remains unclear. Here, we investigate the possibility that microRNAs (miRNAs) are involved in the aspect. Methods: As a model of chronic fluorosis, SD rats received different concentrations of fluoride in their drinking water for 3 or 6 months and SH-SY5Y cells were exposed to fluoride. Literature reviews and bioinformatics analyses were used to predict and real-time PCR to measure the expression of 12 miRNAs; an algorithm-based approach was applied to identify multiply potential target-genes and pathways; the dual-luciferase reporter system to detect the association of miR-132–3p with MAPK1; and fluorescence in situ hybridization to detect miR-132–3p localization. The miR-132–3p inhibitor or mimics or MAPK1 silencing RNA were transfected into cultured cells. Expression of protein components of the MAPK pathway was assessed by immunofluorescence or Western blotting. Results: In the rat hippocampus exposed with high fluoride, ten miRNAs were down-regulated and two up-regulated. Among these, miR-132–3p expression was down-regulated to the greatest extent and MAPK1 level (selected from the 220 genes predicted) was corelated with the alteration of miR-132–3p. Furthermore, miR-132–3p level was declined, whereas the protein levels MAPK pathway components were increased in the rat brains and SH-SY5Y cells exposed to high fluoride. MiR-132–3p up-regulated MAPK1 by binding directly to its 3′-untranslated region. Obviously, miR-132–3p mimics or MAPK1 silencing RNA attenuated the elevated expressions of the proteins components of the MAPK pathway induced by fluorosis in SH-SY5Y cells, whereas an inhibitor of miR-132–3p just played the opposite effect. Conclusion: MiR-132–3p appears to modulate the changes of MAPK signaling pathway in the CNS associated with chronic fluorosis.

Abstract Background Alzheimer’s disease (AD) is responsible for 60–70% of all cases of dementia. On the other hand, the tap water consumed by hundreds of millions of people has been fluoridated to prevent tooth decay. However, little is known about the influence of fluoride on the expression of APP and subsequent changes in learning and memory and neuropathological injury. Our aim here was to determine whether exposure to fluoride aggravates the neuropathological lesions in mice carrying the amyloid precursor protein (APP)/presenilin1 (PS1) double mutation. Methods These transgenic or wide-type (WT) mice received 0.3 ml of a solution of fluoride (0.1 or 1 mg/ml, prepared with NaF) by intragastric administration once each day for 12 weeks. The learning and memory of these animals were assessed with the Morris water maze test. Senile plaques, ionized calcium binding adaptor molecule 1 (Iba-1), and complement component 3 (C3) expression were semi-quantified by immunohistochemical staining; the level of Aβ42 was detected by Aβ42 enzyme-linked immunosorbent assays (ELISAs); the levels of synaptic proteins and enzymes that cleave APP determined by Western blotting; and the malondialdehyde (MDA) content and activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) measured by biochemical procedures. Results The untreated APP mice exhibited a decline in learning and memory after 12 weeks of fluoride treatment, whereas treatment of these some animals with low or high levels of fluoride led to such declines after only 4 or 8 weeks, respectively. Exposure of APP mice to fluoride elevated the number of senile plaques and level of Aβ42, Iba-1, and BACE1, while reducing the level of ADAM10 in their brains. The lower levels of synaptic proteins and enhanced oxidative stress detected in the hippocampus of APP mice were aggravated to fluoride. Conclusions These findings indicate that exposure to fluoride, even at lower concentration, can aggravate the deficit in learning and memory and neuropathological lesions of the mice that express the high level of APP.

Introduction: For investigating the mechanism of brain injury caused by chronic fluorosis, this study was designed to determine whether NRH:quinone oxidoreductase 2 (NQO2) can influence autophagic disruption and oxidative stress induced in the central nervous system exposed to a high level of fluoride. Methods: Sprague–Dawley rats drank tap water containing different concentrations of fluoride for 3 or 6 months. SH-SY5Y cells were either transfected with NQO2 RNA interference or treated with NQO2 inhibitor or activator and at the same time exposed to fluoride. The enrichment of gene signaling pathways related to autophagy was evaluated by Gene Set Enrichment Analysis; expressions of NQO2 and autophagyrelated protein 5 (ATG5), LC3-II and p62, and mammalian target of rapamycin (mTOR) were quantified by Western-blotting or fluorescent staining; and the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) assayed biochemically and reactive oxygen species (ROS) detected by flow cytometry. Results: In the hippocampal CA3 region of rats exposed to high fluoride, the morphological characteristics of neurons were altered; the numbers of autophagosomes in the cytoplasm and the levels of NQO2 increased; the level of p-mTOR was decreased, and the levels of ATG5, LC3-II and p62 were elevated; and genes related to autophagy enriched. In vitro, in addition to similar changes in NQO2, p-mTOR, ATG5, LC3 II, and p62, exposure of SH-SY5Y cells to fluoride enhanced MDA and ROS contents and reduced SOD activity. Inhibition of NQO2 with RNAi or an inhibitor attenuated the disturbance of the autophagic flux and enhanced oxidative stress in these cells exposed to high fluoride. Conclusion: Our findings indicate that NQO2 may be involved in regulating autophagy and oxidative stress and thereby exerts an impact on brain injury caused by chronic fluorosis. [ABSTRACT FROM AUTHOR]

The protective effect of resveratrol (RSV), a stimulator of the silent mating type information regulation 2 homolog (SIRT1), on the neurotoxicity of rat brain induced by chronic fluorosis was investigated. Thirty-two Sprague-Dawley (SD) rats were divided randomly into four groups: (i) a control group; (ii) a RSV treatment group; (iii) a fluoride-exposed group (50 ppm F- in drinking water); and (iv) a fluoride plus RSV group. The experimental period was 7 months. The protein levels of SIRT1 in the cortex and the hippocampus of the rat brains were determined by Western blotting, the SOD activity and the MDA content in the brains by biochemical methods, and the 8-OHdG content by ELISA. The spatial learning ability and the memory of the rats was examined by the Morris Water Maze test. The results showed chronic fluorosis caused changes to the brains of the rats with a reduced SIRT1 protein, elevated MDA and 8-OHdG contents, inhibited SOD activity, and, in addition, decreased learning and memory. Interestingly, RSV pretreatment attenuated the reduced SIRT1, the raised level of oxidative stress, and the lowered ability for learning and memory resulting from the chronic fluorosis. The results indicate that RSV may have a neuroprotective effect on fluoride toxicity. [ABSTRACT FROM AUTHOR]

The levels of DNA methylation were analyzed in the peripheral blood cells of children, with or without skeletal fluorosis, in the area of the coal-burning type of endemic fluorosis in Guizhou Province of China. Whole genome DNA methylation 450 K BeadChip and pyrosequencing were performed, and the main function and pathway of differential methylation genes determined by GO analysis and the KEGG database. The results showed that 15 differential hypermethylated and 22 hypomethylated CpG sites were determined in the children with skeletal fluorosis, which involved 29 known genes, as compared to controls. The majority of the differentially methylated CpG loci resided in the gene body. Further, the GO analysis and the KEGG database for the pathway analysis indicated that these differential genes are mainly involved in synapse maturation, axon, neuron projection membrane, transcription coactivator activity, and the Notch signaling pathway. Pyrosequencing analysis showed that the gene MAML2 was hypomethylated in the children with skeletal fluorosis, which is the same as the chip result. A change concerning genome-wide DNA methylation in the children with skeletal fluorosis was also found. The results suggest that the alteration of the hypomethylated MAML2 gene may play an important role in the occurrence and development of skeletal fluorosis. [ABSTRACT FROM AUTHOR]

The protective role of vitamin E (Vit E) against neurotoxicity induced by fluorosis was investigated by using Sprague-Dawley (SD) rats fed with 50 ppm fluoride in drinking water for 10 months. Spatial learning and memory of rats were measured by the Morris water maze test; the expressions of M1 and M3 muscarinic acetylcholine receptors (mAChRs) at the protein level in the hippocampus and cortex were detected by immunohistochemistry; and the levels of O2 ·- and malondiadehyde (MDA) were evaluated by biochemical methods. The results showed that high fluoride inhibited learning ability and memory of the rats, reduced the protein expressions of both M1 and M3 mAChRs, and elevated the levels of O2 ·- and MDA in the rat brains. Interestingly, the treatment of Vit E prevented the increased production of O2 ·- and MDA in brains of the rats fed with high fluoride. In addition, Vit E attenuated the decreased learning ability and memory of the rats exposed to high fluoride, and the mechanism for this may involve the recovered expression of mAChRs resulting from the use of the antioxidant. [ABSTRACT FROM AUTHOR]