Adolescent ; Adult ; Child ; Female ; Humans ; Male ; Middle Aged ; Myringoplasty ; Risk Factors ; Tympanic Membrane Perforation ; etiology ; surgery ; Young Adult

Objective To investigate the expression and distribution of the downstream substrate of extracellular regulated protein kinase(ERK1/2) pathway, ternary complex factor phospho-Elk-1, in rat brains with chronic fluorosis, and reveal the mechanism of the impaired learning and memory ability caused by chronic fluorosis. Methods Seventy-two SD rats, weighing 100 - 120 g, were randomly divided into 3 groups, 24 in each group (half male and half female). The rats in control group were fed with tap water (fluoride < 0.5 mg/L); low- and high-dose fluoride groups were fed with tap water with different concentrations of NaF(5.0,50.0 mg/L F-, respectively). After 6 months, body weight was weighed, dental fluorosis was determined by observation and urinary fluoride and bone fluoride were detected by fluorine ion-selective electrode; the learning ability of rats was measured by navigation test of Morris water maze, and memory ability by spatial probe test in Morris water maze; the expression and distribution of phospho-Elk-1 in different brain regions were detected by immunohistochemistry method. Results In low- and high-fluoride groups, the body weight of rat[(449.2 ± 77.1), (312.8 ± 89.7)g] was significantly decreased than that of control [(635.5 ± 76.2 )g, all P< 0.05], the varying degrees of dental fluorosis were observed(x2 = 7.83, P<0.05), urinary fluoride[(2.56 ±0.91),(5.73 ±3.14)mg/L] and bone fluoride[(709.2 ± 37.4) ,(1306.3 ± 102.4) mg/kg] were significantly higher than those in controls[(0.92 ± 0.30)mg/L,(348.5 ± 89.2)mg/kg, all P< 0.05]. The escape latency of low- and high-fluoride groups[ (7.4 ± 4.1), (12.2 ± 5.7)s] was longer than that of control [(4.8 ± 2.7 )s, all P < 0.05] and the escape latency in high-fluoride group was significantly longer than that in other groups (all P < 0.05); in spatial probe test, the time of first crossing platform was longer in rats with fluorosis [(4.18 ± 1.10),(5.89 ± 0.56)s] as compared to control[(1.17 ± 0.75)s, all P< 0.05]. Expressions of phospho-Elk-1 in the hippocampus CA1(167.4 ± 8.3,163.2 ± 9.4), CA2(175.7 ± 5.0,183.3 ± 4.2), CA3(165.2 ± 11.6,162.9 ± 4.4), CA4(168.7± 6.9,169.5 ±5.3), fascia dentate (185.2 ±4.0,193.1 ±6.1) and caudate putamen( 181.4 ± 3.8, 179.8 ± 5.5) in low- and high-fluoride groups were higher than those of controls(142.4 ± 8.1,144.9 ± 8.4,143.6 ± 5.8, 116.8 ± 9.1,140.2 ± 7.8,163.1 ± 13.1, all P< 0.05). Conclusion Chronic fluorosis can cause increased expression of phospho-Elk-1 in the hippocampus and caudate putamen region of rat brains, which might be related to the mechanisms of decreased learning and memory ability of rats overexposed to fluoride.

Objective To investigate the changes of reactive oxygen species(ROS) level and mitochondria fission-fusion-balance in cortical neurons of rats with chronic fluorosis and reveal the correlation between these two factors. Methods One hundred and twenty rats were randomly divided into 3 groups(control group, low-dose fluorosis group, high-dose fluorosis group) and 40 rats were in each group according to body weight and the experiments were carried out for 3 months or 6 months. The rats were fed with different concentrations of fluoride (NaF) to establish fluorosis models. Controls were fed with tap water( < 0.5 mg/L), experimental animals in low- or high-dose group were fed with water containing NaF 10.0,50.0 mg/L, respectively. The level of ROS and the morphology in mitochondria fission-fusion balance in neurons of the cortex of rat brains prepared with cortical frozen sections were detected with ROS fluorescent probe and MitoTracker RED probe, respectively. Results Significant differences of the level of ROS and the numbers of abnormal mitochondria in morphology in the cortical neurons were found between 3 groups at the experiment period of 3 month and 6 month(F= 3.07,3.06,3.05,3.07, all P < 0.05). As compared with control group(10.43 ± 5.98,4.12 ± 3.86) at the experiment period of 3 month, the level of ROS and the numbers of abnormal mitochondria in morphology in the cortical neurons were obviously increased in high-dose fluorosis group(25.48 ± 6.09,20.47 ± 6.09, all P < 0.05), whereas no significant changes were found in low-dose fluorosis group(11.67 ± 3.49,6.68 ± 3.48, all P> 0.05). Furthermore, the increases in both ROS level and abnormal numbers of mitochondria were significant observed in the cortical neurons of low-dose fluorosis group (63.02 ± 8.15, 49.33 ± 8.61) and high-dose fluorosis group(65.60 ± 7.40,53.10 ± 6.95) as compared with the control group (25.26 ± 6.41,20.26 ± 6.41) at the experimental period of 6 month (all P < 0.05). The abnormal numbers of mitochondria correlated with ROS level(r = 0.93,0.81, all P < 0.05). Conclusions Taking excessive amount of fluoride results in high level of oxidative stress and impaired the balance of mitochondrial fission-fusion,which is dependent on the feeding times and doses of fluoride. The mechanism of the mitochondrial abnormalities might be associated with the high level of oxidative stress induced by chronic fluorosis.

Objective To observe the expression of mitochondrial fission protein locus Fis1 and ultrastructural changes in the renal cells of rats with chronic fluorosis,and to reveal the mechanism in mitochondrial damage of the renal cells.Methods Sixty SD rats were randomly divided into 3 groups according to sex and body mass(20 in each group):control group,lower fluoride group and higher fluoride group.All the rats were fed with different doses of sodium fluoride in drinking water(0,10 and 50 mg/L,respectively).Six-month later,the expression of Fisl in renal cells was determined by real-time fluorenscence quantitative PCR and immunohistochemistry technology,the mitochondrial morphology of renal cells was observed under transmission electron microscopy (TEM).Results As compared with the control group(28.70 ± 12.41),Fis1 mRNA levels(91.48 + 34.83 and 582.09 ± 184.69) in renal cells of the lower fluoride and the higher fluoride groups were increased(all P ＜ 0.05).As compared with the control group(10.49 ± 7.66),Fisl protein levels(16.33 ± 10.26 and 21.50 ± 5.24) in renal cells of the lower fluoride and the higher fluoride groups showed a trend of increasing,the higher fluoride group was higher than that of the control group(P ＜ 0.05).By TEM,mitochondrial crest in renal cells of the lower fluoride and the higher fluoride groups was vague or disappeared,mitochondrial division section appeared.Conclusions Fluoride is a kind of toxicant that can cause damage to mitochondrion of renal cells,induce the expression of Fis1 in transcriptional and protein level,and lead to the obstacles of mitochondrial fusion-fission and ultrastructural abnormality of mitochondrion,which may play an important role in mechanism of mitochondrial damage in the renal cells of rats with chronic fluorosis.

ObjectiveTo investigate the transcriptional changes of nitochondria fission and fusion gene loci and reactive oxygen species (ROS) level in cortical neurons of rats with chronic fluorosis,and to reveal their roles in mitochondria damage due to chronic fluorosis.MethodsSD rats were fed with different doses of fluoride through drinking water[＜ 0.5(control),10,50 mg/L,respectively] for 3 and 6 months.The level of ROS and mRNA contents of mitochondria fission gene loci Drp1/Fis1 and fusion gene locus Mfn1 in the cortical neurons of rat brains were detected with ROS fluorescent probe and real-time PCR,respectively.ResultsAs compared with control group [10.43 ± 5.98,(3.4 ± 0.6) × 103,(8.8 ± 1.4) × 10,(1.2 ± 0.2) × 102] at the experiment period of 3 months,the level of ROS and mRNA contents of mitochondria fusion gene locus Mfn1 and fission gene loci Drp1/Fis1 in the cortical neurons were obviously increased in the rats fed with 50 mg/L fluoride through drinking water[25.48 ± 6.09,(1.0 ± 0.2) × 1011,(3.0 ± 1.6) × 103,(8.9 ± 3.6) × 102,all P ＜ 0.05],whereas no significant changes were found in the rats fed with 10 mg/L fluoride[11.67 ± 3.49,(3.1 ± 0.3) × 104,(6.7 ± 2.7) × 10,(5.0 ± 0.9) × 10,all P ＞0.05].Furthermore,at 6 months of the experiment the increases in ROS level(63.02 ± 8.15,65.60 ± 7.40) and mRNA contents of mitochondria fission gene loci Drp1/Fis1 [(2.0 ± 0.8) × 106,(4.0 ± 0.6) × 105,(3.8 ± 1.3) × 103,(1.3 ± 0.2) × 103] and the decrease in mitochondrial fusion gene locus Mfn1[(3.0 ± 0.4) × 106、(4.0 ± 0.9) × 104]were observed in the cortical neurons of the rats fed with 10 mg/L and 50 mg/L fluoride as compared with the control group[25.26 ± 6.41,(3.0 ± 0.8) × 109,(5.1 ± 0.8) × 103,(2.8 ± 0.7) × 102,all P ＜ 0.05].Conclusions Excessive intake of fluorine leads to elevated ROS levels,and decreased transcription of mitochondrial fusion gene loci Mfn1,which is positively correlated with the time and dose-exposed to fluoride.The changes of mitochondrial fission and fusion gene loci in the cortical neurons may be related to high level of oxidative stress induced by chronic fluorosis.

Animals ; Female ; Fluorosis, Dental ; blood ; etiology ; Glutathione Peroxidase ; blood ; Male ; Malondialdehyde ; blood ; Myocytes, Cardiac ; pathology ; ultrastructure ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sinoatrial Node ; pathology ; ultrastructure ; Sodium Fluoride ; poisoning ; Superoxide Dismutase ; blood

OBJECTIVETo observe the mitochondrial fragmentation and the expression of mito-fusion 1 gene in the cortical neurons of rats with chronic fluorosis, and to reveal their roles in mitochondria damage to neurons due to chronic fluorosis.

METHODSSD rats were divided randomly into three groups of 20 each (a half females and a half males housed individually in stainless-steel cages), and fed with the different doses of fluoride containing in drinking water (untreated control containing 0 mg/L fluoride, and low-fluoride and high supplemented with 10 and 50 mg/L fluoride, respectively). After 3 or 6 months exposure, the mitochondrial morphology of the neurons in rat brains were observed by transmission electron microscopy (TEM), then the expression of mitochondrial fusion gene, Mfn1, were detected by immunohistochemistry and western-blotting, respectively.

RESULTSDental fluorosis was obvious in the rats exposed to excessive fluoride in their drinking water, that is, (16 rats out of 20) numbers of I° detal fluorosis in the low-fluoride group, and (11 rats out of 20) numbers of I° and (9 rats out of 20) numbers of II° detal fluorosis in the high-fluoride group were observed after 3 months exposure. Moreover, (14 rats out of 20) numbers of I° and (6 rats out of 20) numbers of II° detal fluorosis in the low-fluoride group and (6 rats out of 20) numbers of Io, (13 rats out of 20) numbers of II°, and (1 rats out of 20) numbers of III° detal fluorosis in the high-fluoride group were observed after 6 months exposure. And both of untreated controls without detal fluorosis were also observed. The urinary level of fluoride in the low-fluoride group (3.30 ± 1.18) mg/L and in the high-fluoride group (5.10 ± 0.35) were observed after 3 months exposure (F = 3.18, P < 0.05). Moreover, the urinary level of fluoride in the low-fluoride group (4.16 ± 1.39) mg/L and in the high-fluoride group (5.70 ± 1.70) mg/L were also observed after 6 months exposure (F = 3.17, P < 0.05). The normal mitochondrial morphology of neurons in rats without fluorosis was observed after 3 and 6 months, while the abnormal mitochondrial morphology of neurons with fluorosis was shown, presenting mitochondrial fragmentation with swollen cristae and even the fragmented, shortened or stacked punctuate membranes (section observation of three bullous mitochondrial-mitochondrial fission process) by TEM. As compared with controls (53.0 ± 4.54 and 1.21 ± 0.18) at the experiment period of 3 months, Mif1 protein analysis with immunocytochemical (the numbers of positive cells: 51.09 ± 6.25) and western-blotting (1.22 ± 0.26) were no significant difference for low fluoride group (t = 1.7, 1.1, P > 0.05); Mif1 protein analysis with immunocytochemical (the numbers of positive cells: 59.71 ± 5.64) and western-blotting (1.66 ± 0.20) were significantly increasing for high fluoride group (t = 2.1, 2.1, P < 0.05). As compared with controls (36.43 ± 4.04 and 1.00 ± 0.13) at the experiment period of 6 months, Mif1 protein analysis with immunocytochemical (the numbers of positive cells 20.05 ± 4.55 and 17.10 ± 3.86) and western-blotting (0.64 ± 0.08 and 0.39 ± 0.06) were significantly decreasing for the two fluoride group (t = 2.1, 2.2; 2.2, 2.2 respectively, all P value were < 0.05).

CONCLUSIONSTaking excessive amount of fluoride might result in the mitochondrial fragmentation for the changed expression of Mfn1, and the neurons damage from the chronic fluorosis might be associated with the dysfunction of mitochondrial fusion.

Animals ; Drinking Water ; chemistry ; Female ; Fluoride Poisoning ; metabolism ; pathology ; Fluorosis, Dental ; metabolism ; Male ; Membrane Proteins ; metabolism ; Mitochondria ; pathology ; Mitochondrial Proteins ; metabolism ; Neurons ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the expressions of mRNA and protein of p38, Osx, PI3K, Akt1 in the rats bone with chronic fluorosis.

METHODSDental fluorosis were observed and the fluoride contents in the urine and bone were detected by fluorin-ion selective electrode. The morphologic changes and ultrastructure of rats' bone were observed by light and electronic microscopy. The expressions of protein and mRNA of p38, Osx, PI3K and Akt1 were detected by immunohistochemistry and real-time PCR, respectively. The contents of BALP and BGP in serum were detected by ELISA.

RESULTSThe rates of dental fluorosis in the fluorosis rats were increased, and the fluoride contents in bone and urine of the fluorosis rats were increased compared to the control group, the difference was statistically significant (P < 0.05). The bone trabeculae thickness and density and the thickness of bone cortex in fluorosis rats were remarkably increased, the space of bone trabeculae was reduced, and in accordance with the matching morphometrical indices, the difference was statistically significant (P < 0.05) as compared with the control rats. The contents of BALP [(54.61 ± 2.27) U/L] and BGP [(2.38 ± 0.16) µg/L]in the fluoride groups were higher than those in the control group, the difference was statistically significant (P < 0.05). Ultrastructurally, the broadening of the osseouslacuna was observed. The reduced protuberances of the osteocytes, the unclear organelle structure, pyknosis, karyotheca increasation and edged chromatin were also observed. Compared to the control group, the expressions of protein and its mRNA of p38, Osx, PI3K and Akt1 were higher in the fluorosis rats than those in the control rats, and the difference was statistically significant (P < 0.05). There is no any expression of p38, Osx, PI3K and Akt1 in the osteocytes in fluorosis rats.

CONCLUSIONSThe over-expression of p38, Osx, PI3K and Akt1 in bone tissue of fluorosis rats may relate to the accumulation of fluorine in the body. The bone injury mainly occur in the stage of the differentiation and proliferation. The upregulation of P38MARK signal path and PI3K/Akt1 signal path may be involved in the pathogenesis of bone injury caused by fluoride.

Alkaline Phosphatase ; blood ; Animals ; Bone and Bones ; metabolism ; pathology ; ultrastructure ; Fluoride Poisoning ; metabolism ; pathology ; Fluorides ; metabolism ; urine ; Fluorosis, Dental ; metabolism ; pathology ; Immunohistochemistry ; Microscopy, Electron, Transmission ; Osteocalcin ; blood ; Phosphatidylinositol 3-Kinases ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Sodium Fluoride ; toxicity ; Transcription Factors ; genetics ; metabolism ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism

OBJECTIVETo investigate the changes of mitochondrial distribution in axon/soma and the expression of mitochondrial fission 1 (Fis1) protein in the cortical neurons of rats with chronic fluorosis.

METHODSSixty SD rats were divided into 3 groups (20 each) according to weight hierarchy and fed with different concentrations of fluoride in drinking water (0, 10 and 50 mg/L, respectively) for 6 months. Images of mitochondria and tubulin labeled by immunofluorescence COXIV and tubulin-α were captured with fluorescence microscope. Fis1 protein expression in cortical neurons was analyzed with immunohistochemistry and Western blot. The expression of Fis1 mRNA was detected with real-time PCR.

RESULTSVarying degrees of dental fluorosis and increased fluoride contents in urine were observed in the rats receiving additional fluoride in drinking water. In the cortical neurons of rats fed with 10 mg/L and 50 mg/L fluoride, the numbers of neuronal soma stained with COXIV(34.8 ± 4.7 and 39.3 ± 3.0, respectively), and the expression of Fis1 protein (immunohistochemistry: 54.0 ± 3.6 and 51.3 ± 4.1, respectively; Western blot: 2.9 ± 0.4 and 2.6 ± 0.6, respectively) and mRNA (3773 ± 1292 and 1274 ± 162, respectively) was markedly increased as compared with controls (4.4 ± 2.3, 25.2 ± 2.5, 1.8 ± 0.2 and 277 ± 73) over the experimental period of 6 months.

CONCLUSIONSExcessive intake of fluoride results in an altered mitochondrial distribution in axon and soma in cortical neurons (i.e., the increase in soma and the decrease in axon), increased expression of Fis1 gene and enhanced mitochondrial fission. The altered mitochondrial distribution may be related to the high expression level of Fis1 and a functional disorder of mitochondria.

Animals ; Axons ; pathology ; Cerebral Cortex ; metabolism ; Drinking Water ; adverse effects ; chemistry ; Electron Transport Complex IV ; metabolism ; Female ; Fluorides ; adverse effects ; urine ; Fluorosis, Dental ; etiology ; metabolism ; pathology ; Male ; Mitochondria ; pathology ; Mitochondrial Dynamics ; drug effects ; Mitochondrial Proteins ; genetics ; metabolism ; Neurons ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Tubulin ; metabolism

OBJECTIVETo explore the changes of protein expression of mitochondrial fission gene dynamin-related 1(Drp 1) in the cortical neurons of rats with chronic fluorosis.

METHODSA total of 120 one-month-old SD rats (each weighing approximately 100-120 g at the beginning of the experiment) were randomly divided into three groups, and fed with the different doses of fluoride containing in drinking water (untreated control containing 0 mg/L fluoride, and low-fluoride & high-fluoride supplemented with 10 and 50 mg/L fluoride,respectively). After 3 or 6 months exposure, 20 rats from each group were killed. Then the protein expression of mitochondrial fission gene, Drp1, was detected by immunohistochemistry and western-blotting method.

RESULTSDental fluorosis and urinary fluorosis were obviously found in the rats exposed to fluoride. At the experiment period of 3 months, the numbers of positive cells of Drp1 detected by immunohistochemistry changed. Compared with the control group (36.3 ± 5.8), the changes in low-fluoride group (34.7 ± 4.1) showed no significant difference (t = 1.5, P > 0.05),but the increase in high-fluoride group (45.0 ± 4.7) had statistical significance (t = 8.8, P < 0.05). The western-blotting method had consistent results. Compared with the control group (0.59 ± 0.03), a significant increase of the average topical density in low- fluoride (0.62 ± 0.03) and high-fluoride (0.71 ± 0.02) groups were found (t = 0.02,0.11, P < 0.05). At the experiment period of 6 months, the numbers of positive cells of Drp1 detected by immunohistochemistry significantly changed. Compared with the control group (33.2 ± 4.4), the number in low- fluoride and high-fluoride groups were separately (36.6 ± 3.8) and (39.4 ± 4.2),both increased significantly (t = 3.5,6.3, P < 0.05). Same results could be found in western-blotting method,compared with the control group (0.65 ± 0.06), the average topical density in low- fluoride (0.80 ± 0.09) and high-fluoride (0.76 ± 0.08) groups both increased significantly (t = 0.1,0.1, P < 0.05).

CONCLUSIONSTaking excessive amount of fluoride might result in the changes of expression of Drp1, and the neurons damage from the chronic fluorosis might be associated with the hyperfunction of mitochondrial fusion.

Animals ; Drinking Water ; chemistry ; Dynamins ; genetics ; metabolism ; Fluoride Poisoning ; metabolism ; Fluorides ; urine ; Fluorosis, Dental ; metabolism ; Male ; Mitochondrial Dynamics ; Neurons ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley