fluoride is an ubiquitous element in the environment and has a remarkable prophylactic effect at low concentrations by inhibiting dental caries , while at higher concentrations it causes dental and skeletal fluorosis . endemic fluorosis is prevalent in many parts of the world and causes damage not only to hard tissues of teeth and skeleton , but also to soft tissues , such as brain , liver , kidney , and spinal cord . epidemiological investigations reveal that intelligence quotient ( iq ) of children living in endemic fluorosis areas is lower than that of children living in low fluoride areas [ 37 ] . it has been demonstrated that high concentrations of fluoride can decrease learning ability and memory in some animal experiments [ 8 , 9 ] and result in dysfunctions of the central nervous system ( cns ) [ 10 , 11 ] . as the cases of many chronic degenerative diseases , the increase of reactive oxygen species ( ros ) and lipid peroxidation ( lpo ) has been considered to play an important role in the pathogenesis of chronic fluoride toxicity [ 1214 ] . fluoride administration significantly increases brain lpo level compared with control group in rat , while reduced glutathione ( gsh ) content and superoxide dismutase ( sod ) , glutathione peroxidase ( gpx ) , and glutathione reductase ( gr ) activities decrease markedly in fluoride - treated groups [ 15 , 16 ] . there are significantly negative correlations between fluoride concentrations in brain and gpx activity , gsh level , and positive correlations between fluoride concentrations and thiobarbituric acid reactive substances ( tbarss ) and carbonyl groups . the cns is especially sensitive to free radical oxidative damage as it contains more easily oxidizable fatty acids [ 18 , 19 ] . ros is produced during the respiratory burst of phagocytes , and the regulated generation of ros plays an important role in host defense , oxygen sensing , and signal transduction [ 20 , 21 ] , while excessive production ros promotes cellular injure and tissue damage . macrophages are sources of free radicals , including ros and reactive nitrogen species ( rns ) . microglia are a kind of resident macrophage of the cns and play a vital role in immune surveillance and injury repair [ 22 , 23 ] . microglia activation is a common phenomenon in response to exposure to toxicants , and activated microglia are both phagocytic and potent sources of reactive oxygen and nitrogen intermediates [ 2426 ] . microglia excessive activation also can trigger or exacerbate neurotoxicity by inducing oxidative stress of neurons . nitric oxide ( no ) production results from nitric oxide synthase ( nos ) that catalyze the conversion of l - arginine to l - citrulline and no . at high concentrations , no readily reacts with superoxide anion ( o2 ) , a kind of ros derived from nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase ( nox ) , to produce peroxynitrite ( onoo ) . onoo is able to irreversibly inhibit mitochondrial respiration , react with proteins , lipids , carbohydrates and dna , and cause dna fragmentation and lipid oxidation . a growing number of studies have shown that fluoride can increase the generation of ros and lpo in brain , but it is not known if ros increasing in brain is related with activated microglia at fluoride exposure . in the present study , we treated bv-2 microglia cell line with different concentrations of fluoride and found that bv-2 microglia cells were activated . the results indicated that activating bv-2 microglia cells by fluoride induced oxidative stress , which provides a potential oxidative stress mechanism for fluoride - related brain damage . sodium fluoride ( naf , molecular weight 41.99 ) was procured from sigma chemical ( st . louis , mo , usa ) . all other analytical laboratory chemicals and reagents were obtained from sigma , invitrogen ( carlsbad , ca , usa ) , hyclone ( logan , ut , usa ) .   the immortalized murine microglia cell line , bv-2 , was provided by cell culture center , school of basic medicine , peking union medical college . the bv-2 cells were maintained in dulbecco 's modified eagles medium ( dmem ) that contained 10% fetal bovine serum and antibiotics at 37c in a 5% co2 humified incubator . exponentially growing bv-2 cells at density of 5000 cells in 100  l medium were treated with 0.5120  mg / l naf respectively for 24 , 48 , and 72  h for cell viability assay . / l ) were selected for intracellular sod , gsh , malondialdehyde ( mda ) , ros , o2 , nos , nitrotyrosine ( nt ) measurement and cell medium was collected for no , nos assay . viability and growth patterns of bv-2 cells based on mitochondrial enzyme functions in 96-well plates were determined under naf - treated for 24  h , 48  h , and 72  h by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide ( mtt ) conversion to formazan . briefly , 100  l of mtt solution ( 0.5  mg / ml in the medium ) was added to each well , and the plates were incubated at 37c for additional 4  h. afterwards , the medium containing mtt was removed , and the crystals were dissolved in 150  l of 100% dimethyl sulfoxide ( dmso ) . the cell viability was quantified using a microplate reader ( multiscan ascent , labsystems , finland ) at 570  nm . following naf treatment , cultured microglia were fixed with 4% paraformaldehyde and incubated with the diluted primary antibody ( 1  :  200 ox-42 , microglia marker ) for 20  min at 37c , and secondary antibody sheep anti - mouse igg was added for 20  min at 37c . after washing , the cells were incubated with dab dilution until an observed color change . staining of ox-42 was examined at high - power fields (  400 ) under a standard light microscope . the number of cells as identified by ox-42 antibody was counted in five different fields on each microscope slide . bv-2 cells were cultured in 10  cm diameter dishes and treated with naf as indicated . at the end of treatment , cells were washed 3 times with ice - cold phosphate - buffered saline ( pbs ) , scraped off the dishes with a silicone  policeman  and harvested into eppendorf tubes . then cells were lyzed in pbs by sonication and centrifuged at 15000  g for 5  min at 4c . the intracellular gsh levels and sod activity were measured with improved 5,5-dithiobis-(2-nitrobenzoic acid ) ( dtnb ) method and hydroxylamine assay , respectively , provided by commercial test kits ( keygen biotech . the quantification was based on measuring formation of thiobarbituric acid reactive substances ( tbars ) according to the manufacturer 's protocol . protein concentrations were determined by the method of bradford   to normalize the levels of gsh , sod , and mda . the results were expressed as mg / g protein for gsh , u / mg protein for sod , and nmol / ml for mda . the production of ros was determined by measuring dichlorofluorescein ( dcf ) fluorescence as described by rothe and valet . briefly , bv-2 cells in 24-well culture plates , treated with naf for 24  h , were rinsed and resuspended in serum - free medium containing 10  m dcfh - da . after a further 30  min of incubations at 37c , cells were rinsed twice with ice - cold pbs and harvested by trypsin , and were measured via a facscan flow cytometer ( becton dickinson , usa ) with excitation wave length at 488  nm and emission wave length at 525  nm for ros generation . the fluorescence intensity parallels to the amount of ros formed . briefly , bv-2 cells were harvested after treated with naf for 24  h , washed with serum - free culture medium and incubated with 5  m dhe at 37c for 30  min . then the cells were harvested , washed and resuspended , and were measured via a facscan flow cytometer ( becton dickinson , usa ) with excitation wave length at 300  nm and emission wave length at 610  nm for o2 generation . bv-2 cells were treated with various concentrations of naf for 24  h. the cell culture medium was collected , then centrifuged 1  min at 13000  rpm to remove floating cells and cell debris , and analyzed immediately for nitrite content . the production of no was determined by measurement the nitrite accumulation in the medium using colorimetric assay with griess reagent . nitrite concentrations were determined by comparison with standard solutions of sodium nitrite at optical density 550  nm . then cells were lyzed in pbs by sonication and centrifuged at 15000  g for 5  min at 4c , the resulting supernatants and medium were used as sources of nos , respectively . nos activity was determined by monitoring the formation of [ 3h ] l - citrulline from [ 3h ] l - arginine as the method described previously . the commercial test kit of nos was provided by nanjing jiancheng biological corporation ( nanjing , china ) . bv-2 cells were treated with various concentrations of naf for 24  h , and the intracellular nt was assessed by elisa using monoclonal antibodies and the procedure recommended by the supplier ( abway antibody technology co. , ltd . , the concentration of nt was calculated according to the standard curve of the elisa kits . all experiments were performed at least in triplicate and the values represent mean  sd . differences between groups were statistically analyzed by one - way analysis of variance ( anova ) and positive rates of ox-42 expression were analyzed by the fisher exact test . as shown in figure 1 , fluoride reduced the bv-2 cell viability in 50 , 100 , or 120  mg / l naf groups . at concentrations of 50 , 100 , or 120 mg / l , sodium fluoride caused a significant decrease in cell viability of 57% , 70% , and 72% , respectively , versus control at naf - treated 24  h , 50% , 69% , and 86% , respectively , versus control at naf - treated 48  h , and 51% , 78% , and 92% , respectively , versus control at naf - treated 72  h. integrin m ( ox-42 ) is a cell adhesion molecule that acts as a receptor for cell surface ligands . immunocytochemistry localization with an ox-42 antibody was used as a microglial marker . to confirm the activation of bv-2 in response to naf , ox-42 expression was determined by immunocytochemistry after 24  h incubation with different naf concentrations ( 120  mg / l ) . naf of concentrations from 5 to 20  mg / l can stimulate bv-2 cells to change into activated microglia displaying upregulated ox-42 expression and large bodies compared with control microglia with small bodies and long branch ( figure 2 ) . the number of cells showing ox-42 positive expression markedly increased in 5 , 10 , and 20  mg / l naf - treated bv-2 cells compared with the control . as shown in figure 3 , a marked decrease in sod activities was observed in fluoride - treated bv-2 cells . sod activities decreased significantly by 22.3% , 25.6% , and 34.4% in 1 , 10 , and 50 mg / l naf - treated cells , respectively , compared to the control cells , and there was a significant negative dose - effect relationship ( r = 0.804 , p < 0.01 ) . dcf fluorescence intensities in bv-2 cells increased with fluoride concentrations , with a significant dose - dependent manner ( r = 0.851 , p < 0.01 ) , and the dcf fluorescence intensities in the 50 mg / l naf - treated cells were significantly higher than the control cells ( p < 0.01 ) . mda concentrations also increased in naf - treated cells , and there was a significant positive dose - effect relationship ( r = 0.5 , p < 0.05 ) although there were no statistical differences in naf - treated cells compared with the control cells . however , there was an increase tendency in naf - treated cells compared to the control for the intracellular gsh levels . in addition to ros , rns from activated microglia can be a major cause of oxidative stress . we therefore measured no and nos in bv-2 cells treated with various concentrations of fluoride for 24  h. no level in the culture medium did not increase with fluoride concentrations . however , activities of nos , synthesizing no , significantly increased in 1 , 10 , and 50  mg / l naf - treated bv-2 cell culture medium compared with the control , in a dose - dependent manner ( r = 0.96 , p < 0.01 ) . intracellular nos activities only increased in 10  mg / l naf - treated bv-2 cells compared with the control , see figure 4 . dhe fluorescence intensities in bv-2 cells increased with fluoride concentrations , with a significant dose - dependent manner ( r = 0.943 , p < 0.01 ) , and the dhe fluorescence intensities in the 1 , 10 , and 50 peroxynitrite , the reaction product of no and o2 , can lead to macromolecule damage and formation of nt , a protein adduct , and nt has been as a marker of free radical oxidation of nitric oxide . our study showed that intracellular nt concentrations in bv-2 cells increased significantly following fluoride exposure in 10 and 50  mg / l naf - treated groups compared with the control group , and there also existed a dose - dependent enhancement ( r = 0.603 , p < 0.05 ) , see figure 5 . the redox balance of the cell maintained by oxidants and antioxidants is important to physiological activity and function . disturbance of the natural equilibrium of free radical over generation causes dysfunction and promotes oxidative damage to tissue . although ros have some essential roles in normal cell functions , they are more associated with their pathological effects that ultimately lead to protein and cellular damage as well as cell death [ 32 , 33 ] . ros could attack numerous cell components like membrane lipids , resulting in enhanced lipid peroxidation and cellular toxicity . many studies [ 34 , 35 ] have reported that excessive fluoride exposure can damage the redox balance of the cells in tissues , decrease antioxidant defense capacity in brain , and increase the toxic effects on visceral organs mediated by generation of ros and lipid peroxidation . a close association between chronic fluoride toxicity and increased oxidative stress has been reported in humans and in experimental animals [ 37 , 38 ] . neurotoxicity induced by fluoride had been linked with oxidative stress [ 11 , 16 ] . microglia are the resident immune cells in the brain and readily activated by a variety of foreign substances including environmental toxicants . the beneficial roles of microglia activation include clearance of toxic cell debris and pathogens , induction of innate immune responses , and enhancement of neuronal survival . however , an array of ros and rns produced and secreted from activated microglia has been believed to be involved with neurotoxicity in brain disorders [ 4042 ] . in the present study several in vivo studies have reported microglial proliferation and activation early after various brain insults . thus , it can be hypothesized that oxidative stress of microglia could be one of the causative factors for fluoride induced brain toxicity . once activated , microglia undergo morphological changes as well as phenotypic alterations in some molecules . microglial activation is generally measured by a gradual change in morphology from a quiescent ramified form ( resting state ) to an amoeboid form ( activated state ) , and upregulation of cell surface receptors , such as cd11b ( ox-42 ) . in this study , the majority of naf - treated bv-2 microglial cells displayed an activated morphology and increased the expression of ox-42 receptor , an abundance surface receptor in activated microglia . the increased number of ox-42 microglia observed in this study may be due to the reactive response of microglia to fluoride treatment . to our knowledge , this is the first time to verify that microglia are activated by fluoride . nox is a superoxide producing enzyme system , the major source of ros in microglia [ 4648 ] . superoxide anions ( o2 ) derivated from nox can be catalyzed into the hydrogen peroxide ( h2o2 ) and molecular oxygen by sod . sod accelerates o2 to h2o2 , which can be said as primary defence , as it prevents further generation of free radicals . in the present study , the sod activity significantly decreased in dose - dependent manner and o2 content increased in fluoride - treated bv-2 microglia . superoxide radicals especially cause oxidation of sh groups in protein , and it has been demonstrated that thiolate radicals are removed by gsh . gsh also participates in the reaction that destroys hydrogen peroxide , or organic peroxide , free radicals , and certain foreign compounds . gsh is as a direct free radical scavenger , as a cosubstrate for gpx activity and as a cofactor for many enzymes and forms conjugates in endo- and xeno - biotic reactions . the decrease in gsh level is often observed due to increased utilization in oxidative stress . in contrast , gsh levels in bv-2 microglial cells increased with fluoride concentrations in our present study . oxidative stress is defined as a disturbance in the balance between the production of free radicals and antioxidant defense , and the later is more compensatory . our observed increase of gsh may be a compensation increase to scavenge excessive superoxides and protect cells against oxidative stress . the production of lipid peroxidation , such as mda , was accumulated as a result of membrane disruption , elicited by the oxidation of poly unsaturated fatty acids of the bilayer . the present study found that the mda levels showed an increasing tendency in naf - treated bv-2 cells although there were no significant differences between naf - treated bv-2 cells and control cells . however , ros level in 50  mg / l naf - treated group increased significantly , which indicated that ros could attack membrane lipids and result in lipid peroxidation . no is a compound of special interest released by activated microglia and has been described in vitro situations to be an important mechanism by which microglia cause neuronal death [ 5153 ] . no is produced from l - arginine and molecular oxygen by the action of nos [ 54 , 55 ] in activated microglia and contribute to the oxidative stress accompanying the inflammatory process . nos is the key enzyme for no production and is quantitatively induced in activated glial cells after exposure to stimulators . the neurotoxic effects of no are generally attributed to its reaction with o2 to form onoo , an extremely active oxidizing agent , which can lead to macromolecule damage and form the protein adduct , such as nt , and nt has been as a marker of free radical oxidation of nitric oxide . in the present study , fluoride significantly increased the nos activity in activated bv-2 cells , and which should produce more no . interestingly , in our experiment , no level did not increase after bv-2 cells were exposed to fluoride , that could due to produced no reacted rapidly with o2 to form onoo . indeed , we have observed a significant increase of nt level at 10 and 50  mg / l naf - treated cells , suggesting that neurotoxicity of fluoride is associated with overproduction of nitric oxide and peroxynitrite . therefore , rns , together with ros produced by activated microglia was involved in oxidative stress induced by fluoride in activated bv-2 cells .   in conclusion , a main finding of this study was that microglia bv-2 cells were activated by fluoride , and the increase of ros and rns in microglia was associated with the activation of microglia . the novelty of this work is related to the active role of microglia treated with fluoride . we provided new evidence from in vitro model indicating that fluoride exerts its toxic effects in cns possibly partly ascribed to activating of microglia in vitro , which enhanced oxidative stress induced by ros and rns .