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.

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.

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 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 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 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 observe the expression of neural nicotinic acetylcholine receptor subunit α3 (α3nAChR) and extracellular regulated protein kinases (ERK1/2),c-Jun N-terminal kinase (JNK),p38 kinases of mitogen-activated protein kinase (MAPK) pathway in human neuroblastoma cell line SH-SY5Y overexposed to fluoride,and try to investigate the molecular mechanism of cell damage caused by overexposure of fluoride.Methods The SH-SY5Y cell with low expression of α3nAChR suppressed by silence interference RNA served as α3nAChR silence group;the normal SH-SY5Y cell served as control group,and the effect of silencing of αt3nAChR gene in SHSY5Y was detected by Western blotting and real-time PCR;SH-SY5Y cell was treated with different concentrations of fluoride (0.000,0.005,0.050,0.500,1.000,2.500,5.000 mmol/L),the safe concentration of fluoride in SHSY5Y cell was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay;the SH-SY5Y cell of control group and α3nAChR silence group were treated with 4.000 mmol/L fluoride for 0,4,8,12,24,36,48 h according to the results of MTT assay;the expression of ERK1/2,JNK,p38 kinases of MAPK pathway in SH-SY5Y at protein levels was measured by Western blotting.Results The expression of α3nAChR mRNA (0.04 ± 0.03) and protein (12.0 ± 2.5) in α3nAChR silence group was decreased significantly compared with those of control group (1.00 ± 0.11,100.0 ± 11.3,t =24.58,28.80,all P ＜ 0.05).The viability of SH-SY5Y cell treated with 5.000 mmol/L fluoride (0.53 ± 0.15) was decreased significantly compared with that of SH-SY5Y cell treated with 0.000 mmol/L fluoride (1.05 ± 0.05,P ＜ 0.05).The increased expression of phospho-ERK1/2 was found in α3nAChR silence group and control group incubated with fluoride with time prolonged,and the expression of phospho-ERK1/2 increased significantly at time points 24,36 and 48 h (188.33 ± 7.33,200.00 ± 10.01,213.33 ± 11.55;125.33 ± 5.69,136.00 ± 4.52,155.33 ± 6.51) compared to 0 h in the same groups (100.00 ± 0.00,100.00 ± 0.00,all P ＜ 0.05),and the expression of phospho-ERK1/2 was higher significantly in α3nAChR silence group than those of control group (t =9.26,7.63,5.72,all P ＜ 0.05);no change of expression of total-ERK1/2 in the two groups was found with the passage of time.The gradually increased expression of phospho-JNK was found in α3nAChR silence group and control group,among which,the expression of phospho-JNK in o3nAChR silence group at time points 12,24,36 and 48 h (154.00 ± 6.25,149.00 ± 5.57,156.00 ± 6.08,141.67 ± 2.52) and in control group at 8,12,24,36,48 h (133.33 ± 10.69,173.00 ± 4.00,175.00 ± 11.79,200.67 ± 11.93,200.33 ± 18.58) was compared to those at 0 h in the same groups (100.00 ± 0.00,100.00 ± 0.00),and the difference was significant (all P ＜ 0.05);the higher expression of phospho-JNK was found in α3nAChR silence group other than control group at 8,12,24,36,48 h (t =-4.28,-5.02,-2.89,-8.33,-6.18,all P ＜ 0.05);no change of expression of total-JNK was found in the two groups (P ＞ 0.05).The increased expression of phospho-p38 was detected in control group at time points 24,36 and 48 h (120.33 ± 4.51,122.00 ± 7.55,119.67 ± 7.57) compared to 0 h in the same groups (100.00 ± 0.00,all P ＜ 0.05),and the expression of phospho-p38 was significantly higher than that in α3nAChR silence group at the same time points (93.33 ± 9.61,94.00 ± 5.01,98.33 ± 5.69,t =-4.01,-6.73,-5.59,all P ＜ 0.05);no change of expression of total-p38 was found in the two types of SH-SY5Y cells treated with fluoride (P ＞ 0.05).Conclusion When SH-SY5Y cells are exposed to fluoride;activation of ERK1/2 may be not depend on α3nAChR;α3nAChR may have protected the cell from apoptotic injury caused by activation of JNK pathway,and the activation of p38 may be depend on nAChRα3.

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.

Objective To identify the distribution feature of methylenetetrahydrofolate reductase (M T HFR) gene polymorphism of Buyi ,Dong ,Miao nationality in Guizhou .Methods The MTHFR(677 and 1 298) genotypes of Buyi ,Miao and Dong healthy indi-viduals were determined by TaqMan-MGB probe genotyping method and constructed haplotypes .Results There were significant difference of MTHFR 677C/T genotype and allele frequencies among 3 groups(P<0 .05) ,There was significant difference of geno-type between Buyi and Miao nationality ,and there were significant differences of genotype frequencies in Buyi nationality and Dong and Miao nationality(P<0 .01) .There were no differences of MTHFR 1298A/C genotype frequencies among Buyi ,Dong and Miao nationality(P> 0 .05) .Buyi nationality had the lowest frequency in double wild homozygous type (677CC/1298AA) ,677TT/1298CC double mutation homozygous and 677TT/1298AC combination in above three minorities was not found .There were linkage disequilibrium between 677C/T and 1298A/C in Buyi and Miao nationality .Conclusion The genotypes frequencies of MTHFR 677T T/1298AC are significant differences among different regions and different ethnic groups .

Objective To study the effect of chronic fluorosis on neurobehavioral development,the ability of learning and memory in offspring of rats,and the antagonistic effect of antioxidant Vitamin E (Vit E).Methods According to body weight,forty-five 1-month-old Sprague-Dawley(SD) rats of 30 females and 15 males were divided into three groups by random number table,including control group,fluorosis and Vit E antagonistic groups (15 rats with 10 females and 5 males in every group).Five months after establishing the animal model with chronic fluorosis and Vit E gavage treatments (fluoride ＜ 1,50,50 mg/L,respectively; Vit E 0,0,50 mg/kg,respectively),the rats were mated in 2:1 proportion of female:male in different groups,respectively.The fertility index of female and neurobehavioral development indicators in offspring were observed.Spatial learning and memory of offspring after birth for 30 d were evaluated by using Morris Water Maze test.Results The female fertility index exposed to fluorosis and Vit E were not significantly different as compared to those of control group(all P ＞ 0.05) ; in contrast to control groups[(6.4 + 1.8),(15.1 + 1.7)d],the time that completed the surface righting reflex [(8.1 + 1.4),(7.9 + 1.5)d] and the air righting reflex [(17.7 + 2.3),(17.2 + 1.8)d] were delayed in the offspring in fluorosis and Vit E antagonistic groups(all P ＜ 0.05) ; the completed avoidance precipice reflex and the auditory consternation did not changed significantly(all P ＞ 0.05); In addition,compared with control and Vit E antagonistic groups [(31.74 + 17.78),(34.97 ± 15.44)s,(4.50 ± 2.51),(3.80 ± 1.87)time],the average escape latency and exploration platform at five days were decreased in 30 d offspring of fluorosis group[(42.03 + 16.45)s,(2.20 + 1.62)time].Conclusion Neurobehavioral development as well as learning and memory ability in rat offspring are impaired by long-term exposure to fluoride and Vit E has exhibited an antagonistic effect to the toxicities of fluoride.