To reveal the underling molecular mechanism in brain damage induced by chronic fluorosis, the neurotoxicity and its correlation were investigated by transcriptomics and proteomics.

Expressions of N-methyl-d-aspartic acid receptors (NMDARs) in the brains of rats and primary neurons exposed to high fluoride were investigated. Sprague-Dawley rats were divided randomly into a fluorosis group (50ppm fluoride in the drinking water for 6 months) and controls (<0.5ppm fluoride) and the offspring from these rats sacrificed on postnatal days 1, 7, 14, 21 and 28. The primary cultured neurons from the hippocampus of neonatal rats were treated with 5 and 50ppm fluoride for 48h. NMDAR subunits at protein or mRNA levels were quantified by Western blotting or real-time PCR. The phosphorylated calmodulin-protein kinase II (CaMKII) was determined by Western blotting, concentration of Ca2+ in neurons by laser confocal microscopy and apoptosis by flow cytometry. In the brains of adult rats and pups as well as in primary neurons exposed to high fluoride, the mRNAs encoding GluN1 and GluN2B subunits and the corresponding proteins were elevated, the GluN3A lowered and the GluN2A unchanged. In addition, the level of phosphor-CaMKII was reduced, and Ca2+ influx and apoptosis enhanced in the brains of rats and cultured neurons exposed to high fluoride. The results indicate that such modifications may involve brain damage induced by chronic fluorosis.

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.