Endemic (chronic) fluorosis causes systemic pathological damages,in which the pathogenesis of the disease is complex.Among those relating mechanisms,oxidative stress is a generally accepted theory so far.The comments made in the present paper mainly concern the changes of conventional examinations for oxidative stress,the correlations between oxidative stress and biological membrane lipids,acetylcholine receptors,signal translation systems and apoptosis,and the clinic or experimental investigations on anti-oxidants.It is emphasized that oxidative stress plays an important role in the molecular mechanisms of systemic damages resulted from endemic fluorosis,which may provide theoretical evidence for clinical prevention and treatment,as well as drug development to the disease.

212 Wistar rats were divided randomly into five groups, each of which was fed on one of the following regimes respectively: (1) normal iodine and fluorine, (Ⅱ) normal iodine, 10 ppm fluorine, (Ⅲ) normal iodine, 30 ppm fluorine, (Ⅳ) low iodine, normal fluorine, (Ⅴ) low iodine, 10 ppm fluorine. The total experimental period was seven months. The results showed that severe morphologic and functional damages of the thyroid appeared in the rats drinking water containing 30 ppm fluorine, but only slight abnormal ultrastructural changes of the thyroid cells appeared in the rats drinking water containing 10 ppm fluorine; the rats with iodine deficiency showed proliferative changes of the thyroid; the rats on iodine deficient diet and drinking water containing 10 ppm fluorine showed morphologic and functional damages as well as proliferation. The study suggests that there is a synergistic action of iodine deficiency and fluorine-intoxication on the thyroid.

Objective Through observation of the expression and activity of extracellular regulated protein kinase 5 (ERK5) and its relationship with the learning and memory ability in rats with chronic fluorosis,to further study the pathogenesis of chronic fluorosis in nervous system.Methods Thirty SD rats were divided into 3 groups according to body weight by means of a random number table (10 rats in each group,half male and half female).The rats in control group were fed with free drinking tap water containing less than 0.5 mg/L fluoride (NaF);the rats in low fluoride group with 10.0 mg/L fluoride;the rat in high dose fluoride group with 50.0 mg/L fluoride.After 6months of experiment,rat brain tissue was took,mRNA expression level of ERK5 was detected by real-time fluorescence quantitative PCR (real-time PCR),protein expression level and activity of ERK5 were detected by Western blotting;the learning and memory ability of rats with chronic fluorosis were detected by Morris water maze test.Results The rat in groups exposed to fluoride exhibited different degrees of dental fluorosis and the fluoride content in urine of rats increased gradually with increase of fluoride doses (F =164.10,P ＜ 0.05).The protein levels of phosphor-ERK5 in the control group,low fluoride group and high fluoride group were 0.13 ± 0.03,0.29 ± 0.10and 0.43 ±0.17,respectively,the difference was statistically significant (F=11.96,P＜ 0.05),and low fluoride group and high fluoride group were higher than control group (all P ＜ 0.05).The total protein levels of ERK5 in control group,low fluoride group and high fluoride group were 0.32 ± 0.11,0.37 ± 0.13 and 0.49 ± 0.16,respectively,the difference was statistically significant (F =3.45,P ＜ 0.05),and high fluoride group was higher than control group (P ＜ 0.05).The expression of ERK5 mRNA in rat brains between groups was not significantly different (F =0.81,P ＞ 0.05).The second,third,and forth days of directional navigation experiment,the time of escape latency and the number of crossing the platform between groups were statistically significant (H =28.20,29.90,26.47,27.23,35.34,27.62,all P ＜ 0.01);the fifth day of space exploration experiment,the difference of the time of the first crossing platform and the number of crossing the platform between groups were statistically significant (H =31.41,30.80,all P ＜ 0.01);the protein level of phosphor-ERK5 in brain tissue of rats was negatively correlated with the number of the first crossing platform (r =-0.470,P ＜ 0.01),while positively related to escape latencies at the fifth day of the test (r =0.591,P ＜ 0.01).Conclusion The changes of ERK5 signaling pathway in rat brain tissue caused by chronic fluorosis are found,which are related to the decrease of leaming and memory ability of animals with chronic fluorosis.

Objective To investigate the pathological role of endoplasmic reticulum stress in fluomsisinduced apoptosis in human hepatocellular carcinoma cell line (HepG2).Methods Under stimulation of 1,3,6,9 mmol/L concentrations of NaF in vitro for 24 h,while normal control group was cultured under normal condition,the apoptosis of HepG2 cells was measured by flow cytometry.The endoplasmic reticulum stress markers (glucose regulative proteins 78,94;GRP78,GRP94) and CCAAT/enhancer-binding protein homologous protein (CHOP) in HepG2 cells were measured at both mRNA and protein levels by real-time PCR and Western blotting,respectively.Results After treated with 0,1,3,6,9 mmol/L NaF for 24 h,the apoptosis rate of HepG2 cells was (6.25 ± 1.27)％,(13.48 ± 1.00)％,(24.08 ± 1.88)％,(30.19 ± 3.07)％ and (37.72 ± 4.43)％,respectively,and the difference was statistically significant among groups (F =65.828,P ＜ 0.01).After treated with 3 mmol/L NaF for 24 h,the mRNA level of GRP78,GRP94 and CHOP was (1 172.41 ± 459.60)％,(946.95 ± 635.85)％ and (7 846.97 ± 1 670.01)％,which was increased compared to those of the control groups [(100.00 ± 1.77)％,(100.00 ± 2.08)％,(100.00 ± 0.74)％,t =12.77,4.67,11.50,all P ＜ 0.01].Under the same condition,the protein levels of GRP78 and CHOP were (159.99 ± 67.59)％ and (155.15 ± 94.24)％,which were increased compared to those of the control groups [(100.00 ± 30.68)％,(100.00 ± 41.44)％,t =-3.27,-1.99,all P ＜ 0.05],while GRP94 protein level [(46.40 ± 41.46)％] was decreased compared to that of the control group [(100.00 ± 68.86)％,t =4.02,P ＜ 0.05].Conclusion Endoplasmic reticulum stress may be involved in NaF-induced cell death in HepG2 cells.

Objective To investigate fluoride-induced inflammation and nuclear factor-κB (NF-κB) signaling pathway in cultured human acute monocytic leukemia cells (THP-1).Methods In vitro cultured THP-1 cells were used as a model of microglia.After cultured with different concentrations of [0 (negative control group),10,50,100,500,1 000 and 5 000 μmol/L] sodium fluoride (NaF) for 48 h,the survival of cells was detected by CCK8.THP-1 cells were divided into 3 groups:control group,low dose and high dose fluoride groups according to the results of CCK8 assay,and then treated with different concentrations of sodium fluoride (0,500,5 000 μmol/L) for 48 h,concentrations of inflammatory cytokines,such as Interleukin-lβ (IL-1β) and tumor necrosis factor-α (TNF-α) were measured by enzyme linked immunosorbent assay (ELISA) in THP-1 mononuclear cell culture medium.The protein levels of IκBα,phospho-NF-κB p65 and phospho-IκB-α were detected by Western blotting.Results THP-1 cells were treated with different concentrations of sodium fluoride (500,1 000,5 000 μ mol/L) for 48 h.Fluoride group THP-1 cell survival rate [(73.21 ± 3.67)％,(31.40 ± 4.56)％,(0.40 ± 0.24)％] was lower than that of the negative control group [(100.00 ± 0.00)％,all P ＜ 0.01].Compared to the control groups [(0.36 ± 0.07),(31.07 ± 0.81)ng/L],significant increases of the inflammatory cytokines IL-1β [(1.42 ± 0.79),(19.47 ± 2.90)ng/L] and TNF-α [(61.06 ± 2.20),(172.72 ± 2.29)ng/L] were detected in culture medium in low-fluoride and high fluoride groups,respectively.Interestingly,compared to the control groups [(100.00 ± 5.48)％,(100.00 ± 14.82)％],significant increases of phospho-NF-κB p65 [(113.71 ± 8.99)％,(134.74 ± 1.93)％] and phospho-IκB-α [(152.61 ± 14.16)％,(176.91 ± 7.95)％] were observed in both low-fluoride and high fluoride groups.Meanwhile,the protein level of IκBα in high fluoride group [(63.53 ± 9.67)％] was significantly lower than that of the control group [(100.00 ± 10.99)％,P ＜ 0.01].Furthermore,significant positive correlation was detected between increased IL-1β,TNF-α and phospho-NF-κB p65 (r =0.74,0.72,all P ＜ 0.05).Conclusions Excessive fluoride can induce microglial cells to release inflammatory cytokines and activate nuclear factor-κB signaling pathway.The release of inflammatory cytokines and activation of the signaling pathway may be one of the mechanisms of the damage of the central nervous system caused by sodium fluoride.

Objective To explore the change of hippocampal neurons cholinergic receptor in rats with vascular dementia(VD).Methods VD rat models were established by employing improved method of Pulsinelli's four-vessel occlusion.1 month later,the abilities of learning and memory of VD models were tested by Morris water maze.The activities of acetylcholinesterase (AChE) in plasma and hippocampus were detected by the improved Ellman's colorimetric methods.The expressions of hippocampal nicotinic acetylcholine receptor (nAChR) subunits ?3,?4 and ?7 proteins and mRNA were detected respectinely by Western Blotting and RT-PCR,respectively.The results were compared with sham operation group.Results Compared to the sham operation group,the escape latency of Morris water maze test and the first time crossing platform in VD group were significantly prolonged,the number of crossing platform was decreased (all P

Objective To explore the influences of protein kinase Cβ (PKC3) inhibitor LY333531 on oxidative injury and apoptosis of SH-SY5Y cells induced by fluorosis.Methods The SH-SY5Y cell model of fluorosis was established,and the experiment was divided into three groups:control group [0.0 mmol/L sodium fluoride (NaF) and 0.0 μmol/L LY333531],the fluoride group (0.5 mmol/L NaF and 0.0 μmol/L LY333531),and the PKCβ inhibitor group (0.5 mmol/L NaF and 0.2 μmol/L LY333531),n =3.Flow cytometry was used to detect the changes of reactive oxygen species (ROS) and apoptosis rate,fluorescent probe technique was used to detect mitochondrial membrane potential after each group for 48 h.Results Compared with the control group [(3.32 ± 0.29) × 103,0.60 ± 0.09,(7.58 ± 1.20)％],the level of ROS [(5.99 ± 0.32) × 103] was increased,mitochondrial membrane potential (0.28 ± 0.06) was decreased,and the apoptosis rate [(18.00 ± 2.32)％] was increased in the fluoride group (all P ＜ 0.05);compared with the fluoride group,the level of ROS [(5.12 ± 0.25) × 103] was decreased,mitochondrial membrane potential (0.42 ± 0.03) was increased,and the apoptosis rate [(11.79 ± 0.70)％] was decreased in the PKCβ inhibitor group (all P ＜ 0.05).Conclusions Excess fluoride could cause oxidative damage and apoptosis in cells.PKC3 inhibitor LY333531 has a protective effect in oxidative damage and apoptosis by fluorosis.

Objective To detect the levels of oxidative stress in brain and serum of rats with chronic fluorosis and the antagonistic effects of vitamin E (VitE),and to reveal the role of oxidative stress in brain injury.Methods Thirty healthy SD rats were divided into three groups based on body weight by means of a random number table (10 rats in each group,half male and half female).In the control group,the rats were fed with drinking water containing less than 0.5 mg/L fluoride;in the fluoride group,the rats were fed with high doses of sodium fluoride in drinking water (50.0 mg/L) and the VitE antagonistic group were fed with the same content of fluoride in drinking water as the fluoride group,but adding VitE (50.0 mg/kg) by intragastric administration once a day.All rats were fed with normal diet (6.2 mg/kg).After exposure to fluoride for 10 months,all rats were put to death,dental fluorosis of the rats was examined and the fluoride content in bone was determined by fluoride-ion selective electrode;the activity of superoxide dismutase (SOD) was determined by the xanthine oxidase method and glutathione peroxidase (GSH-Px) by the colorimetric method,the level of malonaldehyde (MDA) by the glucosinolates barbituric acid fluorescence method and the levels of OH-,H2O2 and O-·2 in rat serum and/or brain were detected by the colorimetric method.Results In the rats of the fluoride group,fluoride content in bone was higher as compared to control [bone fluoride:(211.07 ± 48.52) vs.(33.40 ± 9.26) mg/kg,P ＜ 0.01].The activities of SOD and GSH-Px in rat brains of the fluoride group [(20.10 ± 1.98) kU/g,(28.70 ± 19.35) kU/L] were significantly lower than those of controls [(37.05 ± 3.13) kU/g,(59.63 ± 12.83) kU/L,all P ＜ 0.01],the activity of SOD in VitE antagonistic group [(26.27 ± 1.74) kU/g] was higher than the fluoride group (P ＜ 0.01);the activities of SOD and GSH-Px in rat serum of the fluoride group were significantly decreased [(11.55 ± 1.75) kU/L,(79.50 ± 19.18) U/L] than those of controls [(20.79 ± 2.43) kU/L,(170.00 ± 14.68) U/L,all P ＜ 0.01],the activity of SOD in VitE antagonistic group [(17.23 ± 0.68) kU/L] was higher than the fluoride group (P ＜ 0.01).The levels of MDA in rat brain and serum of the fluoride group [(8.84 ± 0.69) μmol/L,(1.46 ± 0.11) nmol/L] were significantly higher than those of controls [(1.27 ± 0.74) μmol/L,(0.83 ± 0.10) nmol/L,all P＜ 0.01],VitE antagonistic groups [(4.51 ± 1.13) μmol/L,(1.29 ± 0.02) nmol/L] were lower than the fluoride groups (all P ＜ 0.01).The levels of OH-,H2O2 and O-·2 in rat brains of the fluoride group [(24.24 ± 1.80) kU/g,(15.28 ± 2.97) mmol/L,(6.53 ± 0.96) U/g] were significantly higher than those of controls [(11.44 ± 1.63) kU/g,(5.28 ± 1.20) mmol/L、,(2.93 ± 0.42) U/g,all P ＜ 0.01],VitE antagonistic groups [(14.43 ± 0.76) kU/g,(8.09 ± 0.55) mmol/L,(4.41 ± 0.49) U/g] were lower than the fluoride groups (all P ＜ 0.01).Conclusions Elevated levels of oxidative stress are found in brain and serum of the rats with chronic fluorosis,which may be a main mechanism of brain injury.VitE may play an important antagonistic role in oxidative damage induced by fluoride toxicity.

Objective To investigate the possible pathological role of mitochondrial apoptosis pathways and its factors in fluorosis-induced apoptosis of human hepatocellular carcinoma cell strain (HepG2).Methods Under the stimulation of 1,3,6 and 9 mmol/L concentrations of NaF in vitro for 24 h (n =5),while normal control group was cultured under normal condition,the cytotoxicity was measured with MTT.The mitochondrial apoptosis inducing factor (AIF) was measured at both mRNA (n =5) and protein levels (n =6),respectively,by real-time PCR and Western blotting.The mitochondrial apoptosis related factors,such as B-cells lymphoma-2 (Bcl-2),Bcl-associated X protein (Bax),cytochrome C,caspase-9 and caspase-3 were measured at protein levels (n =6).Results After treated with 0,1,3,6 and 9 mmol/L NaF for 24 h,the cell absorbance of HepG2 cells was 0.307 ± 0.031,0.333 ± 0.028,0.230 ± 0.011,0.178 ± 0.001 and 0.152 ± 0.003,respectively,and the differences were statistically significant among groups (F =82.224,P ＜ 0.01).After treated with 3 mol/L NaF for 24 h,the mRNA level of AIF was [(153.14 ± 5.41)％] which was increased compared to the control group [(100.00 ± 4.70)％,t =-4.73,P ＜0.05].Under the same condition,the protein levels of AIF,Bcl-2,cytochrome C in cytoplasm,caspase-9 and caspase-3 were (152.16 ± 47.30)％,(171.90 ± 51.52)％,(458.00 ± 19.48)％,(527.17 ± 200.67)％ and (432.70 ±64.27)％,which were increased compared to those of the control groups [(100.00 ± 48.86)％,(100.00 ± 34.44)％,(100.00 ± 116.59)％,(100.00 ± 19.58)％ and (100.00 ± 137.16)％,t =-3.80,-3.96,-15.76,-4.64,-5.06,all P ＜ 0.05],while the protein levels of Bax and cytochrome C in mitochondrion were (24.66 ± 26.04)％,(72.99 ±45.34)％,which were decreased compared to those of the control groups [(100.00 ± 44.01)％,(100.00 ± 34.14)％,t =6.35,0.68,all P ＜ 0.05].Conclusion The mitochondrial apoptosis pathway and related factors may be involved in NaF-induced cell death in HepG2 cells.

Objective To investigate the changes of expression of quinone oxidoreductase-1 (NQO1) and heme oxygenase-1 (HO1) at protein and mRNA levels in the brains of rats with chronic fluorosis,effect on NF-E2-related factor 2/antioxidant response element (Nrf2/ARE) signal pathway,and reveal the mechanism of brain damage induced by the disease.Methods SD rats were randomly divided to two groups of 30 each (half females and half males),e.g.the normal control group (drinking water containing less than 0.5 mg/L of fluorine) and fluoride exposed group (drinking water containing 50.0 mg/L sodium fluoride,NaF).All rats were examined at the 10 months after feeding NaF.Dental fluorosis of rats was observed; the fluoride contents in urine and bone were detected by fluoride-ion selective electrode; protein and mRNA levels of NQO1 and HO1 in brains were detected by Western blotting and quantitative real timePCR,respectively.Results The dental fluorosis was observed,and contents of fluoride in urine [(2.16 ± 0.39)mg/L] and bone [(211.07 ± 40.52)mg/kg] determined in the rats of the fluoride group were higher than those of controls [(1.70 ± 0.24)mg/L,(34.67 ± 11.15)mg/kg,t =2.11,3.23,all P＜ 0.05].The protein expression levels of NQO1 and HO1 in the brains of rats with fluorosis [(255.2 ± 14.3) ％ and (187.2 ± 11.1)％] were also higher than those of controls [(100.0 ± 12.2)％,(100.0 t 8.9)％,t =2.14,2.05,all P ＜ 0.05]; the mRNA levels of NQO1 and HO1 [(210.2 ± 9.8)％ and (154.5 ± 7.4) ％] in the rats of the fluoride group were increased as compared to those of controls [(100.0 ± 10.4)％,(100.0 ± 9.7)％,t =2.33,2.75,all P ＜ 0.05].Conclusion The expression of NQO1 and HO1 in brain of rats with fluorosis are significantly increased,which may be due to the activation of Nrf2/ARE signal pathway and may play a compensative role in enhancing antioxidant ability.