Exposure to fluoride exacerbates the cognitive deficit of diabetic patients living in areas with endemic fluorosis, as well as of rats with type 2 diabetes induced by streptozotocin via a mechanism that may involve excessive activation of the poly(ADP ribose) polymerase-1/P53 pathway.

Epidemiology has shown that fluoride exposure is associated with the occurrence of diabetes. However, whether fluoride affects diabetic encephalopathy is unclear. Elderly diabetic patients in areas with endemic (n = 169) or no fluorosis (108) and controls (85) underwent Montreal Cognitive Assessment. Sprague-Dawley rats receiving streptozotocin and/or different fluoride doses were examined for spatial learning and memory, brain morphology, blood-brain barrier, fasting blood glucose and insulin. Cultured SH-SY5Y cells were treated with 50 mM glucose and/or low- or high-dose fluoride, and P53-knockdown or poly-ADP-ribose polymerase-1 (PARP-1) inhibition. The levels of PARP-1, P53, poly-ADP-ribose (PAR), apoptosis-inducing factor (AIF), and phosphorylated-histone H2A.X (ser139) were measured by Western blotting. Reactive oxygen species (ROS), 8-hydroxydeguanosine (8-OHdG), PARP-1 activity, acetyl-P53, nicotinamide adenine dinucleotide (NAD+), activities of mitochondrial hexokinase1 (HK1) and citrate synthase (CS), mitochondrial membrane potential and apoptosis were assessed biochemically. Cognition of diabetic patients in endemic fluorosis areas was poorer than in other regions. In diabetic rats, fasting blood glucose, insulin resistance and blood-brain barrier permeability were elevated, while spatial learning and memory and Nissl body numbers in neurons declined. In these animals, expression and activity of P53 and PARP-1 and levels of NAD+, PAR, ROS, 8-OHdG, p-histone H2A.X (ser139), AIF and apoptosis content increased; whereas mitochondrial HK1 and CS activities and membrane potential decreased. SH-SY5Y cells exposed to glucose exhibited changes identical to diabetic rats. The changes in diabetic rats and cells treated with glucose were aggravated by fluoride. P53-knockout or PARP-1 inhibition mitigated the effects of glucose with/without low-dose fluoride. Elevation of diabetic encephalopathy was induced by exposure to fluoride and the underlying mechanism may involve overactivation of the PARP-1/P53 pathway.

The influence of NQO2 on the dysfunctional autophagy and oxidative stress induced in the hippocampus of rats and in SH-SY5Y cells by fluoride.

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 autophagy-related 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.

Extract of Ginkgo biloba leaves attenuates neurotoxic damages in rats and SH-SY5Y cells exposed to a high level of fluoride.

BACKGROUND: Potential protection against the neurotoxic damages of high levels of fluoride on rats and SH-SY5Y cells by extract of Ginkgo biloba leaves, as well as underlying mechanisms, were examined. METHODS: The rats were divided randomly into 4 groups, i.e., control, treatment with the extract (100 mg/kg body weight, gavage once daily), treatment with fluoride (50 ppm F- in drinking water) and combined treatment with both; SH-SY5Y cells exposed to fluoride and fluoride in combination with the extract or 4-Amino-1,8-naphthalimide (4-ANI), an inhibitor of poly (ADP-ribose) polymerase-1 (PARP-1). Spatial learning and memory in the rats were assessed employing Morris water maze test; the contents of fluoride in brains and urine by fluoride ion-selective electrode; cytotoxicity of fluoride was by CCK-8 kit; the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the content of malondialdehyde (MDA) by appropriate kits; the level of 8-hydroxydeoxyguanosine (8-OHdG) was by ELISA; the content of ROS and frequency of apoptosis by flow cytometry; the expressions of phospho-histone H2A.X(Ser139), PARP-1, poly (ADP-ribose) (PAR) and Sirtuin-1 (SIRT1) by Western blotting or immunofluorescence. RESULTS: The rats with prolong treatment of fluoride exhibited dental fluorosis, the increased contents of fluoride in brains and urine and the declined ability of learning and memory. In the hippocampus of the rats and SH-SY5Y cells exposed to fluoride, the levels of ROS, MDA, apoptosis, 8-OHdG and the protein expressions of histone H2A.X(Ser139), PARP-1 and PAR were all elevated; the activities of SOD and GSH-Px and the protein expression of SIRT1 reduced. Interestingly, the treatment of Ginkgo biloba extract attenuated these neurotoxic effects on rats and SH-SY5Y cells exposed to fluoride and the treatment of 4-ANI produced a neuroprotective effect against fluoride exposure. CONCLUSION: Ginkgo biloba extract attenuated neurotoxic damages induced by fluoride exposure to rats and SH-SY5Y cells and the underlying mechanism might involve the inhibition of PARP-1 and the promotion of SIRT1.

LiCl attenuates impaired learning and memory of APP/PS1 mice, which in mechanism involves α7 nAChRs and Wnt/ß-catenin pathway.

We examined the mechanism by which lithium chloride (LiCl) attenuates the impaired learning capability and memory function of dual-transgenic APP/PS1 mice. Six- or 12-month-old APP/PS1 and wild-type (WT) mice were randomized into four groups, namely WT, WT+Li (100 mg LiCl/kg body weight, gavage once daily), APP/PS1 and APP/PS1+Li. Primary rat hippocampal neurons were exposed to ß-amyloid peptide oligomers (AßOs), LiCl and/or XAV939 (inhibitor of Wnt/ß-catenin) or transfected with small interfering RNA against the ß-catenin gene. In the cerebral zone of APP/PS1 mice, the level of Aß was increased and those of α7 nicotinic acetylcholine receptors (nAChR), phosphor-GSK3ß (ser9), ß-catenin and cyclin D1 (protein and/or mRNA levels) reduced. Two-month treatment with LiCl at ages of 4 or 10 months weakened all of these effects. Similar expression variations were observed for these proteins in primary neurons exposed to AßOs, and these effects were attenuated by LiCl and aggravated by XAV939. Inhibition of ß-catenin expression lowered the level of α7 nAChR protein in these cells. LiCl attenuates the impaired learning capability and memory function of APP/PS1 mice via a mechanism that might involve elevation of the level of α7 nAChR as a result of altered Wnt/ß-catenin signalling.

Integrated transcriptomic and proteomic analysis indicated that neurotoxicity of rats with chronic fluorosis may be in mechanism involved in the changed cholinergic pathway and oxidative stress.

BACKGROUND: To reveal the underling molecular mechanism in brain damage induced by chronic fluorosis, the neurotoxicity and its correlation were investigated by transcriptomics and proteomics. METHODS: Sprague-Dawley rats were treated with fluoride at different concentrations (0, 5, 50 and 100 ppm, prepared by NaF) for 3 months. Spatial learning and memory were evaluated by Morris water maze test; neuronal morphological change in the hippocampus was observed using Nissl staining; and the level of oxidative stress including reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) were detected by biological methods. The high-throughput transcriptome sequencing (RNA-Seq) and tandem mass tag (TMT) proteomic sequencing were performed to detect the expression of differentially expressed genes and proteins, respectively. RESULTS: The results showed that compared with control group, rats exposed to high-dose fluoride exhibited declined abilities of learning and memory, decreased SOD activity and increased ROS and MDA levels, with lighter colored Nissl bodies. A total of 28 important differentially expressed genes (DEGs) were screened out by transcriptomics. Then, functional enrichment analyses showed that upregulated proteins enriched in cellular transport, while downregulated proteins enriched in synapse-related pathways. Thirteen corresponding DEGs and DAPs (cor-DEGs-DAPs) were identified by differential expressions selected with positively correlated genes/proteins, most of which were related to neurodegenerative changes and oxidative stress response. CONCLUSION: These results provide new omics evidence that rats chronically exposed to high-dose fluoride can induce neurotoxicity in the brains through changes in the cholinergic pathway and oxidative stress.

Protections against toxicity in the brains of rat with chronic fluorosis and primary neurons exposed to fluoride by resveratrol involves nicotinic acetylcholine receptors.

Protection of Resveratrol (RSV) against the neurotoxicity induced by high level of fluoride was investigated. Sprague-Dawley (SD) rats and their offspring, as well as cultures of primary neurons were divided randomly into four groups: untreated (control); treated with 50 mg RSV/kg/ (once daily by gavage) or (20 M in the cultured medium); exposed to 50 ppm F- in drinking water or 4 mmol/l in the cultured medium; and exposed to fluoride then RSV as above. The adult rats were treated for 7 months and the offspring sacrificed at 28 days of age; the cultured neurons for 48 h. For general characterization, dental fluorosis was assessed and the fluoride content of the urine measured (by fluoride-electrode) in the rates and the survival of cultured neurons monitored with the CCK-8 test. The spatial learning and memory of rats were assessed with the Morris water maze test. The levels of α7 and α4 nicotinic acetylcholine receptors (nAChRs) were quantified by Western blotting; and the activities of superoxide dismutase (SOD) and catalase (CAT), and the levels of malondialdehyde (MDA) and H2O2 assayed biochemically. The results showed that chronic fluorosis resulted in the impaired learning and memory in rats and their offspring, and more oxidative stress in both rat brains and cultured neurons, which may be associated the lower levels of α7 and α4 nAChR subunits. Interestingly, RSV attenuated all of these toxic effects by fluorosis, indicating that protection against the neurotoxicity of fluoride by RSV might be in mechanism involved enhancing the expressions of these nAChRs.

Lithium chloride reduced the level of oxidative stress in brains and serums of APP/PS1 double transgenic mice via the regulation of GSK3ß/Nrf2/HO-1 pathway.

Aim: The aim of this study is to investigate whether lithium chloride (LiCl) can regulate glycogen synthase kinase-3ß (GSK3ß)/nuclear factor E2 related factor（Nrf2）/heme oxygenase-1 (HO-1) pathway to reduce the injury of oxidative stress in APP/PS1 double transgenic mice.Materials and Methods: The APP/PS1 double transgenic and wild-type (WT) mice were divided randomly into four groups, i.e. WT, WT + LiCl (LiCl 100 mg/kg by gavage once daily), the transgenic + LiCl and the transgenic groups. The expressions of phosphor-GSK3ß (ser9), Nrf2 and HO-1 at protein levels were detected by Western blotting. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the content of malondialdehyde (MDA) were measured by related detection kits. Nissl bodies in different brain regions were examined by Nissl staining.Results: The decreased protein levels of phosphor-GSK3ß (ser9), Nrf2 and HO-1, the declined activities of SOD and GSH-Px, the increased content of MDA and the decreased Nissl bodies in neurons were observed in the brains or serums of APP/PS1 mice as compared with WT. The treatment with LiCl attenuated these changes in the levels of GSK3ß/Nrf2/HO-1 pathway and oxidative stress as well as Nissl bodies induced by APP/PS1 mutation.Conclusion: LiCl reversed the declined activities of SOD and GSH-Px and the increased content of MDA as well as the decreased Nissl bodies in neurons in the brains or serums of APP/PS1 mice, the mechanism of which may be involved in the down-regulation of the activity of GSK3ß and consequently enhances the expressions of Nrf2 and HO-1.

Exposure to fluoride aggravates the impairment in learning and memory and neuropathological lesions in mice carrying the APP/PS1 double-transgenic mutation.

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 Aß42 was detected by Aß42 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 Aß42, 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.

Changes of learning and memory ability and brain nicotinic receptors of rat offspring with coal burning fluorosis.

The purpose of the investigation is to reveal the mechanism of the decreased ability of learning and memory induced by coal burning fluorosis. Ten offspring SD rats aged 30days, who were born from the mothers with chronic coal burning fluorosis, and ten offspring with same age from the normal mothers as controls were selected. Spatial learning and memory of the rats were evaluated by Morris Water Maze test. Cholinesterase activity was detected by photometric method. The expressions of nicotinic acetylcholine receptors (nAChRs) at protein and mRNA levels were detected by Western blotting and Real-time PCR, respectively. The results showed that in the rat offspring exposed to higher fluoride as compared to controls, the learning and memory ability declined; the cholinesterase activities in the brains were inhibited; the protein levels of alpha3, alpha4 and alpha7 nAChR subunits were decreased which showed certain significant correlations with the declined learning and memory ability; and the mRNA levels of alpha3 and alpha4 nAChRs were decreased, whereas the alpha7 mRNA increased. The data indicated that coal burning fluorosis can induce the decreased ability of learning and memory of rat offspring, in which the mechanism might be connected to the changed nAChRs and cholinesterase.