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 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 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 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 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 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.

Objective To detect the expression of muscarinic acetylcholine receptors (mAChRs) at mRNA and protein levels in the brain of rats with chronic fluorosis and to reveal the role of the receptors in brain injury and learning and memory deficits.Methods Sixty healthy SD rats were divided into two groups (30 rats in each group,half males and half females) by random number table method according to body weight.In the control group,the rats were fed with drinking water containing no more 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).Each group was fed with normal diet (6.2 mg/kg).After being exposed to fluoride for 10 months,behavioral performance was measured with Morris water maze,including the escape latency time and the numbers of crossing platforms.After being sacrificed,rat brains were taken and weighted.M1 and M3 subunits at mRNA and protein levels were detected by real-time PCR and Western blotting,respectively; the correlation between protein levels of the receptor subunits and the ability of learning and memory was analyzed.Results In fluoride group,the escape latency time [(21.68 ± 2.90)s] was significantly longer than that of control group [(6.14 ± 1.71)s,t =0.289,P ＜ 0.05]; and the number of crossing platforms [(11.62 ± 2.26)times] was significantly decreased as compared to that of control group [(19.00 ± 3.69)times,t =0.352,P ＜ 0.05].Furthermore,the mRNA expression [(17.07 ± 6.89)％,(12.25 ± 5.03)％] and the protein levels [(71.07 ± 6.89)％,(32.25 ± 4.66)％] of M1 and M3 receptors in rat brains were significantly lower as compared to those of controls [(100.00 ± 3.00)％,(100.00 ± 2.15)％ and (100.00 ± 9.01)％,(100.00 ± 10.33)％,t =0.210,0.157,0.095,0.296,all P ＜ 0.05].The escape latency and M1,M3 protein levels were negatively correlated (r =-0.683,-0.700,all P ＜0.05),and the number of space exploration and M1,M3 protein levels were positively correlated (r =0.867,0.837,all P ＜ 0.05).Conclusion Declined expression of mAChRs at mRNA and protein levels have been detected in the brain of rats with chronic fluorosis,which may be one of the main mechanism concerning the learning and memory deficits.

Objective To observe the changes of learning and memory ability and detect the expression of muscarinic acetylcholine receptor (mAChR,M receptor) at mRNA and protein levels in brains of offspring rats with chronic fluorosis,and to reveal the mechanism of the central nervous system damage.Methods Forty healthy SD rats were divided into two groups (20 in each group,half male and half female) by random number table according to body weight.In the control group,the rats were fed with drinking water containing no more than 0.5 mg/L fluoride;in the fluoride group,the rats were fed with high dose of sodium fluoride in drinking water (50.0 mg/L fluoride).Each group was fed with normal diet (6.2 mg/kg fluoride).After exposed to fluoride for 6 months,each group was mated,and brains of newborn offspring rats aged 1,7,14,21 and 28 days were taken,and expression of M1 and M3 receptors at mRNA and protein levels were analyzed by real-time PCR and Western blotting,respectively.Behavioral changes were measured by Morris water maze test at the 28 days after birth.The correlations between protein levels of M1 and M3 receptors and the ability of learning and memory at the 28 days after birth were analyzed.Results In fluoride group of the offspring rats at 28 days after birth,the escape latency time [(35.61 ± 9.00)s] was significantly longer than that in control group [(8.46 ± 3.09)s,P ＜ 0.05],while the numbers of crossing the platforms and the time of staying the platforms [(5.00 ± 2.90)times,(16.66 ± 2.79)s] were significantly decreased as compared to that of control group [(15.17 ± 3.66)times,(22.51 ± 2.66)s,all P ＜ 0.05].Furthermore,the mRNA expression and the protein levels of M1 and M3 receptors in rat brain at each phase in fluoride group were significantly decreased as compared to controls [M1 mRNA in control groups:(100.00 ± 11.00)％,(100.00 ± 17.57)％,(100.00 ± 9.14)％,(100.00 ± 7.52)％,(100.00 ± 15.78)％;M1 mRNA in fluoride groups:(20.47 ± 8.07)％,(14.00 ± 4.53)％,(16.57 ± 7.62)％,(25.56 ± 12.78)％,(16.27 ± 4.82)％;M3 mRNA in control groups:(100.00 ± 16.30)％,(100.00 ± 14.40)％,(100.00 ± 7.20)％,(100.00 ± 14.31)％,(100.00 ± 13.16)％;M3 mRNA in fluoride groups:(29.17 ± 8.00)％,(12.77 ± 2.22)％,(26.40 ± 7.20)％,(15.74 ± 3.55)％,(28.14 ± 7.53)％;M1 protein in control groups:(100.00 ± 2.24)％,(100.00 ± 8.30)％,(100.00 ± 4.61)％,(100.00 ± 13.78)％,(100.00 ± 11.72)％;M1 protein in fluoride groups:(20.47 ± 8.07)％,(14.00 ± 4.53)％,(16.57 ± 7.62)％,(25.56 ± 12.78)％,(16.27 ± 4.82)％;M3 protein in control groups:(100.00 ± 16.30)％,(100.00 ± 14.40)％,(100.00 ± 7.20)％,(100.00 ± 14.31)％,(100.00 ± 13.16)％;M3 protein in fluoride groups:(29.17 ± 8.00)％,(12.77 ± 2.22)％,(26.40 ± 7.20)％,(15.74 ± 3.55)％,(28.14 ± 7.53)％,P ＜ 0.05 or ＜ 0.01].The escape latency and M1,M3 receptors protein levels were negatively correlated (r =-0.827,-0.742,all P ＜ 0.05),and the number of space exploration and M1,M3 receptors protein levels were positively correlated (r =0.843,0.806,all P ＜ 0.05).Conclusion The expression of M receptor at protein and mRNA levels in offspring rat brains of different ages are significantly declined,which might be one of the mechanism of the decreased ability of learning and memory induced by fluoride toxicity.

Objective To observe the expression of NF-E2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein-1 (Keap1) at mRNA and protein levels in the brain of rats with chronic fluorosis and to reveal the mechanism of brain damage induced by the factors.Methods Ninety SD rats were divided into three groups (30 rats in each group,half male half female) by the random number table method according to body weight.The control group was fed with normal tap-water,high-fluoride group with 50 mg/L fluoride (NaF) added in drinking water;and the high-fluoride plus vitamin E (Vit E) group with the same dose of NaF as the high-fluoride group,but giving 5 mg/kg Vit E by intragastric administration.The experiment period was 10 months.The fluoride contents in urine and bone were detected by fluoride-ion selective electrode.The protein and mRNA levels of Nrf2 and Keap1 in brain of rats were detected by Western blotting and quantitative real time PCR,respectively.The activity of superoxid dismutas (SOD) and the content of lipid peroxidation (MDA) were measured by biochemistry methods.Results Dental fluorosis was detected in high-fluoride group.The differences of fluoride contents in urine and bone were statistically significant between groups (F =6.87,182.87,all P ＜ 0.05).The urine fluoride [(2.16 ± 0.39),(2.07 ± 0.15)mg/L] and bone fluoride [(211.07 ± 40.52),(82.09 ± 28.60)mg/kg] in the high-fluoride and high-fluoride plus Vit E groups were higher than those of the control group [(1.70 ± 0.24)mg/L,(34.67 ± 11.15)mg/kg,all P ＜ 0.05].The differences of mRNA and protein levels of Nrf2 and Keap1 in brains of rats,SOD activity,MDA content were statistically significant between groups (F =654.33,432.87,447.45,398.88,68.34,68.34,all P ＜ 0.05).The mRNA levels of Nrf2 and Keap1 [(320.18 ± 6.83)％,(267.37 t 7.22)％] were increased compared to those of control group [(100.00 ±3.00)％,(100.00 ± 2.75)％,all P ＜ 0.05];the protein levels of Nrf2 and Keap1 [(283.28 ± 6.89)％,(196.32 ± 5.57)％]were also raised compared with those of control group [(100.00 ± 8.71)％,(100.00 ± 9.23)％,all P ＜ 0.05];the activity of SOD [(22.10 ± 2.10)μ,mol/kg] in brain of rats in fluoride group was significantly lower and the content of MDA [(8.63 ± 0.77) μmol/kg] was higher than those of control group [(35.05 ± 2.98),(1.25 ± 0.64) μmol/kg,all P ＜ 0.05].In high-fluoride plus Vit E group,the mRNA levels of Nrf2 and Keap1 [(243.23 ± 5.34)％,(180.54 ± 4.48)％] and the protein levels of Nrf2 and Keap1 [(210.88 ± 4.79)％,(150.68 ± 6.49)％] were lower than those of high-fluoride group (all P ＜ 0.05);the activity of SOD [(26.33 ± 1.84)μmol/kg] was significantly higher and the content of MDA [(4.88 ± 0.84)μmol/kg] was lower than that of high-fluoride group (all P ＜ 0.05).Correlation analysis showed that increased levels of Nrf2 and Keap1 were positively correlated with the level of MDA in rat brain (r =0.69,0.33,all P ＜ 0.05),but negatively correlated with the activity of SOD (r =-0.78,-0.80,all P ＜0.05).Conclusion The expression of Nrf2 and Keap1 in brain of rats with fluorosis is significantly increased and positively correlated with the content of fluoride in bone and the level of oxidative stress,whereas vit E can attenuate these abnormal changes induced by fluoride,which might be one of the mechanisms of brain damage of the disease.